Interferon-beta fusion proteins and uses

ABSTRACT

A fusion polypeptide is described having the amino acid sequence X—Y—Z, or portion thereof, comprising the amino acid sequence of a glycosylated interferon-beta (X); Y is an optional linker moiety; and Z is a polypeptide comprising at least a portion of a polypeptide other than glycosylated interferon-beta. It is preferred that X is human interferon-beta-1a. Mutants of interferon-beta-1a are also described.

RELATED APPLICATIONS

[0001] This is a continuation of PCT/US99/24200, filed on Oct. 15, 1999as a continuation-in-part of prior U.S. Provisional Ser. No. 60/104,491filed Oct. 16, 1998 and U.S. Provisional Ser. No. 60/120,237 filed Feb.16, 1999. The teachings of the earlier-filed patent applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Use of polypeptides and proteins for the systemic treatment ofspecific diseases is now well accepted in medical practice. The rolethat these substances play in therapy is so important that many researchactivities are being directed towards the synthesis of large quantitiesby recombinant DNA technology. Many of these polypeptides are endogenousmolecules which are very potent and specific in eliciting theirbiological actions.

[0003] A major factor limiting the usefulness of these proteinaceoussubstances for their intended application is that, when givenparenterally, they are eliminated from the body within a short time.This can occur as a result of metabolism by proteases or by clearanceusing normal pathways for protein elimination such as by filtration inthe kidneys. The problems associated with these routes of administrationof proteins are well known in the pharmaceutical industry, and variousstrategies are being used in attempts to solve them.

[0004] A peptide family, which has been the focus of much clinical work,and efforts to improve its administration and bio-assimilation, is theinterferons. Interferons have been tested in a variety of clinicaldisease states. The use of human interferon beta, one member of thatfamily, is best established in the treatment of multiple sclerosis. Twoforms of recombinant interferon beta, have recently been licensed inEurope and the U.S. for treatment of this disease. One form isinterferon-beta-1a (trademarked, sold as AVONEX ®, mfg. Biogen, Inc.,Cambridge, Mass.) and hereinafter, “interferon-beta-1a” or “IFN-beta-1a”or “IFN-β-1a” or “interferon-β-1a”, used interchangeably. The other formis interferon-beta-1b (trademarked and sold as BETASERON®, Berlex,Richmond Calif.), hereinafter, “interferon-beta-1b”. Interferon beta-1ais produced in mammalian cells using the natural human gene sequence andis glycosylated, whereas interferon beta-1b is produced in E. colibacteria using a modified human gene sequence that contains agenetically engineered cysteine-to-serine substitution at amino acidposition 17 and is non-glycosylated.

[0005] Previously, several of us have directly compared the relative invitro potencies of interferon-beta-1a and interferon beta 1b infunctional assays and showed that the specific activity ofinterferon-beta-1a is approximately 10-fold greater than the specificactivity of interferon-beta-1b (Runkel et al., 1998, Pharm. Res. 15:641-649). From studies designed to identify the structural basis forthese activity differences, we identified glycosylation as the only oneof the known structural differences between the products that affectedthe specific activity. The effect of the carbohydrate was largelymanifested through its stabilizing role on structure. The stabilizingeffect of the carbohydrate was evident in thermal denaturationexperiments and SEC analysis. Lack of glycosylation was also correlatedwith an increase in aggregation and an increased sensitivity to thermaldenaturation. Enzymatic removal of the carbohydrate frominterferon-beta-1a with PNGase F caused extensive precipitation of thedeglycosylated product.

[0006] These studies indicate that, despite the conservation in sequencebetween interferon-beta-1a and interferon-beta-1b, they are distinctbiochemical entities and therefore much of what is known aboutinterferon-beta-1b cannot be applied to interferon-beta-1a, and viceversa.

SUMMARY OF THE INVENTION

[0007] We have exploited the advantages of glycosylated interferon-betarelative to non-glycosylated forms. In particular, we have developed aninterferon-beta-1a composition with increased activity relative tointerferon-beta-1b and that also has the salutory properties of fusionproteins in general with no effective loss in activity as compared tointerferon-beta-1a forms that are not fusion proteins. Thus, ifmodifications are made in such a way that the products (interferon-beta1a fusion proteins) retain all or most of their biological activities,the following properties may result: altered pharmacokinetics andpharmacodynamics leading to increased half-life and alterations intissue distribution (e.g, ability to stay in the vasculature for longerperiods of time) Such a formulation is a substantial advance in thepharmaceutical and medical arts and would make a significantcontribution to the management of various diseases in which interferonhas some utility, such as multiple sclerosis, fibrosis, and otherinflammatory or autoimmune diseases, cancers, hepatitis and other viraldiseases and diseases characterized by neovascularization. Inparticular, the ability to remain for longer periods of time in thevasculature allows the interferon-beta-1a to be used to inhibitangiogenesis and potentially to cross the blood-brain barrier.

[0008] In particular, the invention relates to an isolated polypeptidehaving the amino acid sequence X—Y—Z, wherein X is a polypeptide havingthe amino acid sequence, or portion thereof, consisting of the aminoacid sequence of interferon beta; Y is an optional linker moiety; and Zis a polypeptide comprising at least a portion of a polypeptide otherthan interferon beta. Optional moiety Y and required moiety Z may belinked to either the N- or C-terminus of inteferon beta (X). Preferably,X is human interferon-beta-1a. In the preferred embodiments, Z is atleast a portion of a constant region of an immunoglobulin and can bederived from an immunoglobulin of the class selected from IgM, IgG, IgD,IgA, and IgE. If the class is IgG, then it is selected from one of IgG1,IgG2, IgG3 and IgG4. The constant region of human IgM and IgE contain 4constant regions (CHI, (hinge), CH2, CH3 and CH4, whereas the constantregion of human IgG, IgA and IgD contain 3 constant regions (CH1,(hinge), CH2 and CH3. In the most preferred fusion proteins of theinvention, the constant region contains at least the hinge, CH2 and CH3domains. In other embodiments, moiety Z is at least a portion of apolypeptide that contains immunoglobulin-like domains. Examples of suchother polypeptides include CD1, CD2, CD4, and members of class I andclass II major histocompatability antigens.

[0009] Another embodiment of the invention is a fusion protein having anamino terminal region consisting of the amino acid sequence ofinterferon beta or a portion thereof and having a carboxy terminalregion comprising at least a portion of a protein other than interferonbeta. The carboxy portion is preferably at least a portion of a constantregion of an immunoglobulin derived from an immunoglobulin of the classselected from IgM, IgG, IgD, IgA, and IgE. In the most preferred fusionproteins, the constant region contains at least the hinge, CH2 and CH3domains.

[0010] Another embodiment of the invention is a fusion protein whoseinterferon beta moiety (e.g., X in the formula above) has been mutatedto provide for muteins with selectively enhanced antiviral and/orantiproliferative activity or other advantageous properties relative tonon-mutated forms of interferon-beta-1a.

[0011] Yet another embodiment of the invention is an isolated DNAencoding for the fusion proteins described above. The invention alsopertains to a recombinant DNA comprising an isolated DNA encoding thefusion proteins described above and an expression control sequence,wherein the expression control sequence is operatively linked to theDNA. The scope of the invention also includes host cells transformedwith the recombinant DNA sequences of the invention.

[0012] The invention further pertains to a method of producing arecombinant polypeptide comprising: providing a population of host cellsaccording to the invention; growing the population of cells underconditions whereby the polypeptide encoded by the recombinant DNA isexpressed; and isolating the expressed polypeptide.

[0013] A further aspect of the invention is a interferon-beta 1a fusionprotein comprising interferon-beta-1a and additional polypeptide withwhich it is not natively associated, in substantially purified form, thefusion having an antiviral activity that is about equal to theanti-viral activity of interferon-beta-1a lacking the additionalpolypeptide.

[0014] Yet another aspect of the invention is a pharmaceuticalcomposition comprising a therapeutically effective amount of aninterferon-beta-1a fusion protein.

[0015] Yet another aspect of the invention is a method of inhibitingangiogenesis and neovascularization using the polypeptides of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1 cDNA and deduced amino acid sequence of a histidinetagged-interferon-beta fusion (also called “his IFN-beta” or“His₆-tagged”). The full DNA and protein sequences of the hisIFN-beta-1a are shown. The cleaved VCAM-1 signal sequence leaves 3 aminoterminal residues (SerGlyGly) upstream of the histidine tag (HiS₆,positions 4-9). The enterokinase linker sequence (AspAspAspAspLys) isseparate from the histidine tag by a spacer (positions 10-12,SerSerGly). The natural IFN-beta-1a protein sequence spans positions(Met18-Asn183).

[0017]FIG. 2. cDNA and deduced amino acid sequence for aninterferon-beta-1a/Fc fusion. The full DNA and protein sequences of thehuman IFN-beta-1 a/mouse Fc are shown. The human IFN-beta-1a proteinsequences span amino acid residues 1-166 (DNA sequences 1-498). Theenterokinase linker sequence spans amino acid residues 167-171 (DNAsequences 499-513). The murine IgG2a heavy chain protein sequence spansresidues 172-399 (DNA sequences 514-437).

[0018]FIG. 3. Binding of alanine substituted interferon-beta mutants toa dimeric fusion protein comprised of the extracellular domain of thetype I interferon receptor chain, IFNAR2/Fc. The binding affinities ofthe alanine substituted IFN mutants (A1-E) for the IFNAR2 receptor chainwere determined as described in Example 1 (subsection D). The histogrampresents their binding affinities in this assay relative to wild typehis-IFN-beta (% w.t.). The % w. t. values were calculated as the(affinity of wild type his-IFN-beta)/(affinity of mutant IFN-beta)×100.The % w. t. (x) for multiple assays (n=3) and an average % w.t. (x) forthe experimental set are shown. Mutants A2, AB 1, AB2, and E did notbind IFNAR2/Fc at concentrations 500-fold higher than the w.t.his-IFN-beta EC 50(*).

[0019]FIG. 4. Binding of alanine substituted interferon-beta mutants tothe type I interferon cell surface receptor complexes (“IFNAR1/2complex”) expressed on Daudi Burkitt's lymphoma cells. The receptorbinding properties of the alanine substitution mutants (A1-E) weredetermined using a FACS based, cell surface receptor binding assay asdescribed in Example 1 (subsection D). The histogram presents theirreceptor binding affinities in this assay relative to wild typehis-IFN-beta (% w.t.). The % w. t. for each mutant was calculated as(affinity of the w.t. his-IFN-beta)/(affinity of mutant IFN-beta)×100.The % w.t. values ( ) from multiple assays under the histogram and anaverage of the % w.t. values for the experimental set (x) are shown.

[0020]FIG. 5. Antiviral activities of alanine substitutedinterferon-beta mutants The antiviral activities of the alaninesubstitution mutants (A1-E) were determined on human A549 cellschallenged with EMC virus as described in Example 1 (subsection E). Thehistogram presents their activities in this assay relative to wild typehis-IFN-beta (% w.t.). The % w. t. was calculated as the inverse of theconcentration of mutant IFN-beta (50% cpe)/concentration of w.t.his-IFN-beta (50% cpe)×100. The % w.t ( ) for multiple assays and theaverage of the experimental data set (x) are shown.

[0021]FIG. 6. Antiproliferative activities of alanine substitutedinterferon-beta mutants The antiproliferation activity of the alaninesubstitution mutants (A1-E) were determined on Daudi Burkitt's lymphomacells as described in Example 1 (subsection E). The histogram presentstheir activities in this assay relative to wild type his-IFN-beta (%w.t). The % w. t. was calculated as the (w.t. his-IFN-beta concentration(50% growth inhibition)/mutant IFN-beta concentration (50% growthinhibition)×100. The % w.t ( ) for multiple assays and the average ofthe experimental data set (x) are shown

[0022]FIG. 7. Relative antiviral and antiproliferative activities ofalanine substituted interferon-beta mutants. The relative activities ofalanine substitution mutants (A1-E) in the antiviral (x axis) andantiproliferation (y axis) assays were compared. The average percentwild type his-IFN-beta (% w. t.(x)) presented in FIGS. 5 and 6 were usedfor this comparison. Those mutants with a coordinate loss/gain inactivity would fall on or very near the vertical line. Those mutantswhich have a disproportionate loss/gain in antiviral orantiproliferation activities would fall significantly off the diagonalline (DE1, D, C1). Significance was determined from consideration ofstandard deviations inherent in the average % w. t. values used.

[0023]FIG. 8. Antiviral Activity of interferon-beta-1a/Ig fusion.

[0024] The activity of interferon-beta-1a (used as AVONEX®) orinterferon-beta-1a/murine Ig2a fusion at the concentrations indicated onthe X axis were assessed in antiviral assays using human lung carcinoma(A549) cells challenged with EMC virus. Following a two day incubationwith virus, viable cells were stained with MTT, the plates were read at450 nm, and the absorbance which is reflective of cell viability isshown on the Y axis. The standard deviations are shown as error bars.The concentration of interferon-beta-1a (used as AVONEX® bulkintermediate) which offered (50% maximum OD450) and therefore 50% viralkilling (the “50% cytopathic effect”) was about 0.4 pM and the 50%cytopathic effect for interferon-beta-1a fusion was about 0.15 pM.

[0025]FIG. 9. Measurements of interferon-beta antiviral activity in theplasma of mice treated with interferon-beta-1a/Fc fusion orinterferon-beta-1a.

[0026] Mice are injected iv with either 50,000 Units ofinterferon-beta-1a (used as AVONEX® bulk intermediate) or 50,000 Unitsof interferon-beta-1a/Fc fusion. Blood from these mice is obtained viaretro-orbital bleeds at various times after interferon injection asindicated on the X axis. There are at least 3 mice bled at each timepoint, and plasma is prepared and frozen until the time interferon-betaactivity is evaluated in antiviral assays using human lung carcinoma(A549) cells challenged with encephalomyocarditis virus. Viable cellswere stained with a solution of MTT, the plates were read at 450 nm, todetermine the absorbance which is reflective of cell viability andinterferon-beta activity. Standard curves were generated for each plateusing interferon-beta-1a as AVONEX® and used to determine the amount ofinterferon-beta activity in each sample. Data from the individualanimals are shown.

[0027]FIG. 10. Full DNA and protein sequences of the open reading framesof a direct fusion of human IFN beta and human IgG1Fc (ZL5107)

[0028]FIG. 11. Full DNA and protein sequences of the open reading frameof a fusion protein consisting of human IFN beta/G4S linker/human IgG1FC(ZL6206).

[0029]FIG. 12. Schematic representation of overall cloning andexpression strategy.

DETAILED DESCRIPTION

[0030] All references cited in the Detailed Description are incorporatedherein by references, unless stipulated otherwise. The following termsare used herein:

[0031] I. Definitions:

[0032] Interferon—An “interferon” (also referred to as “IFN”) is asmall, species-specific, single chain polypeptide, produced by mammaliancells in response to exposure to a variety of inducers such as viruses,polypeptides, mitogens and the like. The most preferred interferon usedin the invention is glycosylated, human, interferon-beta that isglycosylated at residue 80 (Asn 80) and that is preferably derived viarecombinant DNA technologies. This preferred glycosylated interferonbeta is called “interferon-beta-1a” (or “IFN-beta-1a” or “IFN-∃-1a” oror “interferon beta 1a” or “interferon-beta-1a” or “interferon-∃-1a”,all used interchangeably). The term “interferon-beta-1a” is alsointended to encompass all mutant forms (i.e., Example 1) provided thatthe mutants are also glycosylated at the Asn 80 residue.

[0033] Recombinant DNA methods for producing proteins, includinginterferons are known. See for example, U.S. Pat. Nos. 4,399,216,5,149,636, 5,179,017 (Axel et al) and 4,470,461 (Kaufman).

[0034] Preferred interferon-beta-1a polynucleotides that may be used inthe present methods of the invention are derived from the wild-typeinterferon beta gene sequences of various vertebrates, preferablymammals and are obtained using methods that are well-known to thosehaving ordinary skill in the art such as the methods described in thefollowing U.S. Patents: U.S. Pat. No. 5,641,656 (issued Jun. 24, 1997:DNA encoding avian type I interferon proprotein and mature avian type Iinterferon), U.S. Pat. No. 5,605,688 (Feb. 25, 1997-recombinant dog andhorse type I interferons); U.S. Pat. No. 5,231,176 (Jul. 27, 1993, DNAmolecule encoding a human leukocyte interferon); ); U.S. Pat. No.5,071,761 (Dec. 10, 1991, DNA sequence coding for sub-sequences of humanlymphoblastoid interferons LyIFN-alpha-2 and LyIFN-alpha-3); U.S. Pat.No. 4,970,161 (Nov. 13, 1990, DNA sequence coding for humaninterferon-gamma); U.S. Pat. No. 4,738,931 (Apr. 19, 1988, DNAcontaining a human interferon beta gene); U.S. Pat. No. 4,695,543 (Sep.22, 1987, human alpha-interferon Gx-1 gene and U.S. Pat. No. 4,456,748(Jun. 26, 1984, DNA encoding sub-sequences of different, naturally,occurring leukocyte interferons).

[0035] Mutants of interferon-beta-1a may be used in accordance with thisinvention. Mutations are developed using conventional methods ofdirected mutagenesis, known to those of ordinary skill in the art.Moreover, the invention provides for functionally equivalentinterferon-beta-1a polynucleotides that encode for functionallyequivalent interferon-beta-1a polypeptides.

[0036] A first polynucleotide encoding interferon-beta-1a is“functionally equivalent” compared with a second polynucleotide encodinginterferon-beta-1a if it satisfies at least one of the followingconditions:

[0037] (a): the “functional equivalent” is a first polynucleotide thathybridizes to the second polynucleotide under standard hybridizationconditions and/or is degenerate to the first polynucleotide sequence.Most preferably, it encodes a mutant interferon having the [therapeutic]activity of an interferon-beta-1a;

[0038] (b) the “functional equivalent” is a first polynucleotide thatcodes on expression for an amino acid sequence encoded by the secondpolynucleotide.

[0039] In summary, the term “interferon” includes, but is not limitedto, the agents listed above as well as their functional equivalents. Asused herein, the term “functional equivalent” therefore refers to aninterferon-beta-1a protein or a polynucleotide encoding theinterferon-beta-1a protein that has the same or an improved beneficialeffect on the mammalian recipient as the interferon of which it isdeemed a functional equivalent. As will be appreciated by one ofordinary skill in the art, a functionally equivalent protein can beproduced by recombinant techniques, e.g., by expressing a “functionallyequivalent DNA”. Accordingly, the instant invention embracesinterferon-beta-1a proteins encoded by naturally-occurring DNAs, as wellas by non-naturally-occurring DNAs which encode the same protein asencoded by the naturally-occurring DNA. Due to the degeneracy of thenucleotide coding sequences, other polynucleotides may be used to encodeinterferon-beta-1a. These include all, or portions of the abovesequences which are altered by the substitution of different codons thatencode the same amino acid residue within the sequence, thus producing asilent change. Such altered sequences are regarded as equivalents ofthese sequences. For example, Phe (F) is coded for by two codons, TTC orTTT, Tyr (Y) is coded for by TAC or TAT and His (H) is coded for by CACor CAT. On the other hand, Trp (W) is coded for by a single codon, TGG.Accordingly, it will be appreciated that for a given DNA sequenceencoding a particular interferon there will be many DNA degeneratesequences that will code for it. These degenerate DNA sequences areconsidered within the scope of this invention.

[0040] “fusion”—refers to a co-linear, covalent linkage of two or moreproteins or fragments thereof via their individual peptide backbones,most preferably through genetic expression of a polynucleotide moleculeencoding those proteins. It is preferred that the proteins or fragmentsthereof are from different sources. Thus, preferred fusion proteinsinclude an interferon-beta-1a protein or fragment covalently linked to asecond moiety that is not an interferon. Specifically, an“interferon-beta/Ig fusion” is a protein comprising an interferon betamolecule of the invention (i.e., interferon-beta-1a), or fragmentthereof whose N-terminus or C-terminus is linked to an N-terminus of animmunoglobulin chain wherein a portion of the N-terminus of theimmunoglobulin is replaced with the interferon beta. A species ofinterferon-beta/Ig fusion is an “interferon-beta/Fc fusion” which is aprotein comprising an interferon beta molecule of the invention (i.e.,interferon-beta-1a) linked to at least a part of the constant domain ofan immunoglobulin. A preferred Fc fusion comprises an interferon betamolecule of the invention linked to a fragment of an antibody containingthe C terminal domain of the heavy immunoglobulin chains.

[0041] Also, the term “fusion protein” means an interferon beta proteinchemically linked via a mono- or hetero-functional molecule to a secondmoiety that is not an interferon beta protein and is made de novo frompurified protein as described below.

[0042] “Recombinant,” as used herein, means that a protein is derivedfrom recombinant, mammalian expression systems. Protein expressed inmost bacterial cultures, e.g., E. coli, will be free of glycan so theseexpression systems are not preferred. Protein expressed in yeast mayhave oligosaccharide structures that are different from that expressedin mammalian cells.

[0043] “Biologically active,” as used throughout the specification as acharacteristic of interferon-beta 1a, means that a particular moleculeshares sufficient amino acid sequence homology with the embodiments ofthe present invention disclosed herein to be capable of antiviralactivity as measured in an in vitro antiviral assay of the type shown inExample 1, as described below.

[0044] A “therapeutic composition” as used herein is defined ascomprising the proteins of the invention and other physiologicallycompatible ingredients. The therapeutic composition may containexcipients such as water, minerals and carriers such as protein.

[0045] “amino acid”—a monomeric unit of a peptide, polypeptide, orprotein. There are twenty amino acids found in naturally occurringpeptides, polypeptides and proteins, all of which are L-isomers. Theterm also includes analogs of the amino acids and D-isomers of theprotein amino acids and their analogs.

[0046] A “derivatized” amino acid is a natural or nonnatural amino acidin which the normally occurring side chain or end group (or sugar moietyin the case of interferon-beta-1a) is modified by chemical reaction.Such modifications include, for example, gamma-carboxylation,beta-carboxylation, pegylation, sulfation, sulfonation, phosphorylation,amidization, esterification, N-acetylation, carbobenzylation,tosylation, and other modifications known in the art. A “derivatizedpolypeptide” is a polypeptide containing one or more derivatized aminoacids and/or one or more derivatized sugars, if the polypeptide isglycosylated.

[0047] “protein”—any polymer consisting essentially of any of the 20amino acids. Although “polypeptide” is often used in reference torelatively large polypeptides, and “peptide” is often used in referenceto small polypeptides, usage of these terms in the art overlaps and isvaried. The term “protein” as used herein refers to peptides, proteinsand polypeptides, unless otherwise noted.

[0048] “functional equivalent” of an amino acid residue is an amino acidhaving similar physico-chemical properties as the amino acid residuethat was replaced by the functional equivalent.

[0049] “mutant”—any change in the genetic material of an organism, inparticular any change (i.e., deletion, substitution, addition, oralteration) in a wild-type polynucleotide sequence or any change in awild-type protein. The term “mutein” is used interchangeably with“mutant”.

[0050] “wild-type”—the naturally-occurring polynucleotide sequence of anexon of a protein, or a portion thereof, or protein sequence, or portionthereof, respectively, as it normally exists in vivo.

[0051] “standard hybridization conditions”—salt and temperatureconditions substantially equivalent to 0.5× SSC to about 5× SSC and 65°C. for both hybridization and wash. The term “standard hybridizationconditions” as used herein is therefore an operational definition andencompasses a range of hybridization conditions. Higher stringencyconditions may, for example, include hybridizing with plaque screenbuffer (0.2% polyvinylpyrrolidone, 0.2% Ficoll 400; 0.2% bovine serumalbumin, 50 mM Tris-HCl (pH 7.5); 1 M NaCl; 0.1% sodium pyrophosphate;1% SDS); 10% dextran sulfate, and 100 μg/ml denatured, sonicated salmonsperm DNA at 65° C. for 12-20 hours, and washing with 75 mM NaCl/7.5 mMsodium citrate (0.5× SSC)/1% SDS at 65° C. Lower stringency conditionsmay, for example, include hybridizing with plaque screen buffer, 10%dextran sulfate and 110 μg/ml denatured, sonicated salmon sperm DNA at55° C. for 12-20 hours, and washing with 300 mM NaCl/30 mM sodiumcitrate (2.0× SSC)/1% SDS at 55° C. See also Current Protocols inMolecular Biology, John Wiley & Sons, Inc. New York, Sections6.3.1-6.3.6, (1989).

[0052] “expression control sequence”—a sequence of polynucleotides thatcontrols and regulates expression of genes when operatively linked tothose genes.

[0053] “operatively linked”—a polynucleotide sequence (DNA, RNA) isoperatively linked to an expression control sequence when the expressioncontrol sequence controls and regulates the transcription andtranslation of that polynucleotide sequence. The term “operativelylinked” includes having an appropriate start signal (e.g., ATG) in frontof the polynucleotide sequence to be expressed and maintaining thecorrect reading frame to permit expression of the polynucleotidesequence under the control of the expression control sequence andproduction of the desired polypeptide encoded by the polynucleotidesequence.

[0054] “expression vector”—a polynucleotide, such as a DNA plasmid orphage (among other common examples) which allows expression of at leastone gene when the expression vector is introduced into a host cell. Thevector may, or may not, be able to replicate in a cell.

[0055] “Isolated” (used interchangeably with “substantially pure”)—whenapplied to nucleic acid i.e., polynucleotide sequences, that encodepolypeptides, means an RNA or DNA polynucleotide, portion of genomicpolynucleotide, cDNA or synthetic polynucleotide which, by virtue of itsorigin or manipulation: (i) is not associated with all of apolynucleotide with which it is associated in nature (e.g., is presentin a host cell as an expression vector, or a portion thereof); or (ii)is linked to a nucleic acid or other chemical moiety other than that towhich it is linked in nature; or (iii) does not occur in nature. By“isolated” it is further meant a polynucleotide sequence that is: (i)amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) chemically synthesized; (iii) recombinantly produced by cloning; or(iv) purified, as by cleavage and gel separation.

[0056] Thus, “substantially pure nucleic acid” is a nucleic acid whichis not immediately contiguous with one or both of the coding sequenceswith which it is normally contiguous in the naturally occurring genomeof the organism from which the nucleic acid is derived. Substantiallypure DNA also includes a recombinant DNA which is part of a hybrid geneencoding additional sequences.

[0057] “Isolated” (used interchangeably with “substantially pure”)—whenapplied to polypeptides means a polypeptide or a portion thereof which,by virtue of its origin or manipulation: (i) is present in a host cellas the expression product of a portion of an expression vector; or (ii)is linked to a protein or other chemical moiety other than that to whichit is linked in nature; or (iii) does not occur in nature. By “isolated”it is further meant a protein that is: (i) chemically synthesized; or(ii) expressed in a host cell and purified away from associatedproteins. The term generally means a polypeptide that has been separatedfrom other proteins and nucleic acids with which it naturally occurs.Preferably, the polypeptide is also separated from substances such asantibodies or gel matrices (polyacrylamide) which are used to purify it.

[0058] “heterologous promoter”—as used herein is a promoter which is notnaturally associated with a gene or a purified nucleic acid.

[0059] “Homologous”—as used herein is synonymous with the term“identity” and refers to the sequence similarity between twopolypeptides, molecules or between two nucleic acids. When a position inboth of the two compared sequences is occupied by the same base or aminoacid monomer subunit (for instance, if a position in each of the two DNAmolecules is occupied by adenine, or a position in each of twopolypeptides is occupied by a lysine), then the respective molecules arehomologous at that position. The percentage homology between twosequences is a function of the number of matching or homologouspositions shared by the two sequences divided by the number of positionscompared×100. For instance, if 6 of 10 of the positions in two sequencesare matched or are homologous, then the two sequences are 60%homologous. By way of example, the DNA sequences CTGACT and CAGGTT share50% homology (3 of the 6 total positions are matched). Generally, acomparison is made when two sequences are aligned to give maximumhomology. Such alignment can be provided using, for instance, the methodof Needleman et al., J. Mol Biol. 48: 443-453 (1970), implementedconveniently by computer programs such as the Align program (DNAstar,Inc.). Homologous sequences share identical or similar amino acidresidues, where similar residues are conservative substitutions for, or“allowed point mutations” of, corresponding amino acid residues in analigned reference sequence. In this regard, a “conservativesubstitution” of a residue in a reference sequence are thosesubstitutions that are physically or functionally similar to thecorresponding reference residues, e.g., that have a similar size, shape,electric charge, chemical properties, including the ability to formcovalent or hydrogen bonds, or the like. Particularly preferredconservative substitutions are those fulfilling the criteria defined foran “accepted point mutation” in Dayhoff et al., 5: Atlas of ProteinSequence and Structure, 5: Suppl. 3, chapter 22: 354-352, Nat. Biomed.Res. Foundation, Washington, D.C. (1978).

[0060] The terms “polynucleotide sequence” and “nucleotide sequence” arealso used interchangeably herein.

[0061] The terms “neovascularization” and “angiogenesis” mean, in theirbroadest sense, the recruitment of new blood vessels. In particular,“angiogenesis” also refers to the recruitment of new blood vessels at atumor site.

[0062] “IFNAR2”, “IFNAR1”, “IFNAR1/2” refer to the proteins knows tocompose the cell surface type I interferon receptor. The extracellularportion (ectodomain) portion of the IFNAR2 chain alone can bindinterferon alpha or beta.

[0063] Practice of the present invention will employ, unless indicatedotherwise, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA, protein chemistry, andimmunology, which are within the skill of the art. Such techniques aredescribed in the literature. See, for example, Molecular Cloning: ALaboratory Manual, 2nd edition. (Sambrook, Fritsch and Maniatis, eds.),Cold Spring Harbor Laboratory Press, 1989; DNA Cloning, Volumes I and II(D. N. Glover, ed), 1985; Oligonucleotide Synthesis, (M. J. Gait, ed.),1984; U.S. Pat. No. 4,683,195 (Mullis et al.,); Nucleic AcidHybridization (B. D. Hames and S. J. Higgins, eds.), 1984; Transcriptionand Translation (B. D. Hames and S. J. Higgins, eds.), 1984; Culture ofAnimal Cells (R. I. Freshney, ed). Alan R. Liss, Inc., 1987; ImmobilizedCells and Enzymes, IRL Press, 1986; A Practical Guide to MolecularCloning (B. Perbal), 1984; Methods in Enzymology, Volumes 154 and 155(Wu et al., eds), Academic Press, New York; Gene Transfer Vectors forMammalian Cells (J. H. Miller and M. P. Calos, eds.), 1987, Cold SpringHarbor Laboratory; Immunochemical Methods in Cell and Molecular Biology(Mayer and Walker, eds.), Academic Press, London, 1987; Handbook ofExperiment Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds.), 1986; Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, 1986.

[0064] II. Production and Expression of Fusion Proteins

[0065] The present invention relates to a system for the generation ofinterferon-beta-1 a fusion proteins. In particular, the presentinvention relates to these proteins as well as the recombinant DNAmolecules utilized in their production.

[0066] The production of the polypeptides of this invention may beachieved by a variety of methods known in the art. For example, fulllength interferon-beta-1a or truncated forms of interferon-beta-1a maybe produced by known recombinant DNA techniques using cDNAs (see below).

[0067] A gene which encodes the desired interferon-beta-1a polypeptidemay be designed based on the amino acid sequence of the desiredpolypeptide. Standard methods may then be applied to synthesize thegene. For example, the amino acid sequence may be used to construct aback-translated gene. A DNA oligomer containing a nucleotide sequencecapable of coding for interferon-beta-1a may be made in a single step.Alternately, several smaller oligonucleotides coding for portions of thedesired interferon-beta-1a may be synthesized and then ligated together.Preferably, the DNA sequence encoding the interferon-beta-1a moiety willbe made as several separate oligonucleotides which are subsequentlylinked together. (See Example 2). The individual oligonucleotidestypically contain 5′ or 3′ overhangs for complementarity assembly.

[0068] Once assembled, preferred genes will be characterized bysequences that are recognized by restriction endonucleases (includingunique restriction sites for direct assembly into a cloning orexpression vector), preferred codons taking into consideration the hostexpression system to be used (preferably a mammalian cell), and asequence which, when transcribed, produces a stable, efficientlytranslated RNA. Proper assembly may be confirmed by nucleotidesequencing, restriction mapping, and expression of a biologically activepolypeptide in a suitable host.

[0069] Mammalian interferon beta cDNAs may be isolated by using anappropriate human interferon beta DNA sequence as a probe for screeninga particular mammalian cDNA library by cross-species hybridization.Mammalian interferon beta used in the present invention includes, by wayof example, primate, human, murine, canine, feline, bovine, equine andporcine interferon beta. Mammalian interferon beta can be obtained bycross species hybridization, using a single stranded cDNA derived fromthe human interferon beta DNA sequence as a hybridization probe toisolate interferon beta cDNAs from mammalian cDNA libraries. Among themethods that can be used for isolating and cloning interferon genesequences are those methods found in the U.S. Patents summarized above.Of particular relevance, however, are the teachings of U.S. Pat. No.4,738,931 (Apr. 19, 1988) describing DNA containing a human interferonbeta gene.

[0070] The present invention also related to recombinant DNA moleculescomprising the aforementioned DNA sequences. The recombinant DNAmolecules of this invention are capable of directing expression of thepolypeptides of the invention in hosts transformed therewith. A DNAsequence encoding a fusion polypeptide of the invention must beoperatively linked to an expression control sequence for suchexpression. To provide for adequate transcription of the recombinantconstructs of the invention, a suitable promoter/enhancer sequence maypreferably be incorporated into the recombinant vector, provided thatthe promoter/expression control sequence is capable of drivingtranscription of a nucleotide sequence encoding a glycosylatedinterferon beta. Promoters which may be used to control the expressionof the immunoglobulin-based fusion protein include, but are not limitedto, the SV40 early promoter region (Benoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:144-1445), the regulatory sequences of the metallothioninegene (Brinster et al., 1982, Nature 296:39-42); plant expression vectorscomprising the nopaline synthetase promoter region (Herrera-Estrella etal., Nature 303:209-213) or the cauliflower mosaic virus 35S RNApromoter (Gardner, et al., 1981, Nucl. Acids Res. 9:2871), and thepromoter for the photosynthetic enzyme ribulose biphosphate carboxylase(Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephophatase promoter, and the following animal transcriptional controlregions, which exhibit tissue specificity and have been utilized intransgenic animals: elastase I gene control region which is active inpancreatic cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al.,1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987,Hepatology 7:425-515); insulin gene enhancers or promoters which areactive in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122);immunoglobulin gene enhancers or promoters which are active in lymphoidcells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985,Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444); the cytomegalovirus early promoter and enhancer regions(Boshart et al., 1985, Cell 41:521-530); mouse mammary tumor viruscontrol region which is active in testicular, breast, lymphoid and mastcells (Leder et al., 1986, Cell 45:485-495); albumin gene control regionwhich is active in liver (Pinkert et al., 1987, Genes and Devel.1:268-276); alpha-fetoprotein gene control region which is active inliver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer etal., 1987, Science 235:53-58); alpha 1-antitrypsin gene control regionwhich is active in the liver (Kelsey et al, 1987, Genes and Devel.1:161-171); beta-globin gene control region which is active in myeloidcells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986,Cell 46:89-94; myelin basic protein gene control region which is activein oligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); myosin light chain-2 gene control region which is active inskeletal muscle (Sani, 1985, Nature 314:283-286); and gonadotropicreleasing hormone gene control region which is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378). Prokaryoticexpression systems such as the LAC, or beta-lactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731) are not presently preferred inasmuch as the expressedinterferon beta will not be glycosylated. Nevertheless, prokaryoticexpression systems that will allow glycosylation of interferon beta ineither prokaryotic or eukaryotic hosts are encompassed within the scopeof the invention.

[0071] The expression vectors which can be used include, but are notlimited to, the following vectors or their derivatives: human or animalviruses such as vaccinia virus, adenovirus or retroviral based vectors;insect viruses such as baculovirus; yeast vectors; bacteriophage vectors(e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.Specifically, useful expression vectors for the preferred eukaryotichosts include vectors comprising expression control sequences from SV40,bovine papillomavirus, cytomegalovirus. Further, within each specificexpression vector, various sites may be selected for insertion of theseDNA sequences. These sites are usually designated by the restrictionendonuclease which cuts them. They are well-recognized by those of skillin the art. It will be appreciated that a given expression vector usefulin this invention need not have a restriction endonuclease site forinsertion of the chosen DNA fragment. Instead, the vector may be joinedby the fragment by alternate means.

[0072] The expression vector, and the site chosen for insertion of aselected DNA fragment and operative linking to an expression controlsequence, is determined by a variety of factors such as: the number ofsites susceptible to a particular restriction enzyme, the size of thepolypeptide, how easily the polypeptide is proteolytically degraded, andthe like. The choice of a vector and insertion site for a given DNA isdetermined by a balance of these factors.

[0073] The recombinant constructs of the invention may be introducedinto host cells which are capable of expressing the fusion protein usingany method known in the art, including transformation (for example,using DEAE-dextran or calcium phosphate techniques), transfection,microinjection, infection, cell gun, and electroporation. Any host celltype may be utilized provided that the fusion protein recombinantnucleic acid sequences would be adequately transcribed into mRNA in thatcell type and the cell can glycosylate the protein. In addition, therecombinant nucleic acid constructs of the invention may be used tocreate non-human transgenic animals capable of producing theimmunoglobulin based fusion protein. In preferred embodiments of theinvention, the host cell is a mammalian cell, such as a COS or CHO cell.

[0074] Successful incorporation of these polynucleotide constructs intoa given expression vector may be identified by three general approaches:(a) DNA-DNA hybridization, (b) presence or absence of “marker” genefunctions, and (c) expression of inserted sequences. In the firstapproach, the presence of the interferon-beta-1a gene inserted in anexpression vector can be detected by DNA-DNA hybridization using probescomprising sequences that are homologous to the inserted fusion proteingene. In the second approach, the recombinant vector/host system can beidentified and selected based upon the presence or absence of certain“marker” gene functions (e.g., thymidine kinase activity, resistance toantibiotics such as G418, transformation phenotype, occlusion bodyformation in baculovirus, etc.) caused by the insertion of foreign genesin the vector. For example, if the polynucleotide is inserted so as tointerrupt a marker gene sequence of the vector, recombinants containingthe insert can be identified by the absence of the marker gene function.In the third approach, recombinant expression vectors can be identifiedby assaying the foreign gene product expressed by the recombinantvector. Such assays can be based, for example, on the physical orfunctional properties of the gene product in bioassay systems.

[0075] It will be appreciated that not all host/expression vectorcombinations will function with equal efficiency in expressing DNAsequences encoding the polypeptides of this invention. However, aparticular selection of a host-expression vector combination may be madeby those of skill in the art after due consideration of the principlesset forth herein without departing from the scope of the invention.

[0076] The preferred embodiment of the invention contemplates fusionproteins and DNA sequences coding for them. These fusion proteins havean amino-terminal region characterized by the amino acid sequence ofinterferon-beta-1a and a carboxy-terminal region comprising a domain ofa protein other than interferon-beta-1a. A preferred generic formula forsuch a protein is a protein having a primary amino acid sequence X—Y—Z,wherein X is a polypeptide having the amino acid sequence, or portionthereof, consisting of the amino acid sequence of human interferon beta;Y is an optional linker moiety; and Z is a polypeptide comprising atleast a portion of a polypeptide other than human interferon beta. Inone embodiment, moiety Z can be a portion of a polypeptide that containsimmunoglobulin-like domains. Examples of such other polypeptides includeCD1, CD2, CD4, and members of class I and class II majorhistocompatability antigens. See U.S. 5,565,335 (Capon et al.) forexamples of such polypeptides.

[0077] Moiety Z can include, for instance, a plurality of histidineresidues or, preferably, the Fc region of an immunoglobulin, “Fc”defined herein as a fragment of an antibody containing the C terminaldomain of the heavy immunoglobulin chains.

[0078] In the most preferred fusion proteins, the interferon-beta-1apolypeptide is fused via its C-terminus to at least a portion of the Fcregion of an immunoglobulin. The interferon-beta-1a forms theamino-terminal portion, and the Fc region forms the carboxy terminalportion. In these fusion proteins, the Fc region is preferably limitedto the constant domain hinge region and the CH2 and CH3 domains. The Fcregion in these fusions can also be limited to a portion of the hingeregion, the portion being capable of forming intermolecular disulfidebridges, and the CH2 and CH3 domains, or functional equivalents thereof.These constant regions may be derived from any mammalian source(preferably human) and may be derived from any appropriate class and/orisotype, including IgA, IgD, IgM, IgE and IgG1, IgG2, IgG3 and IgG4.

[0079] Recombinant nucleic acid molecules which encode the Ig fusionsmay be obtained by any method known in the art (Maniatis et al., 1982,Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.) or obtained from publicly available clones.Methods for the preparation of genes which encode the heavy or lightchain constant regions of immunoglobulins are taught, for example, byRobinson, R. et al., PCT Application, Publication No. WO87-02671. ThecDNA sequence encoding the interferon molecule or fragment may bedirectly joined to the cDNA encoding the heavy Ig contant regions or maybe joined via a linker sequence. In further embodiments of theinvention, a recombinant vector system may be created to accommodatesequences encoding interferon beta in the correct reading frame with asynthetic hinge region. Additionally, it may be desirable to include, aspart of the recombinant vector system, nucleic acids corresponding tothe 3′ flanking region of an immunoglobulin gene including RNAcleavage/polyadenylation sites and downstream sequences. Furthermore, itmay be desirable to engineer a signal sequence upstream of theimmunoglobulin fusion protein-encoding sequences to facilitate thesecretion of the fused molecule from a cell transformed with therecombinant vector.

[0080] The present invention provides for dimeric fusion molecules aswell as monomeric or multimeric molecules comprising fusion proteins.Such multimers may be generated by using those Fc regions, or portionsthereof, of Ig molecules which are usually multivalent such as IgMpentamers or IgA dimers. It is understood that a J chain polypeptide maybe needed to form and stabilize IgM pentamers and IgA dimers.Alternatively, multimers of interferon-beta-1a fusion proteins may beformed using a protein with an affinity for the Fc region of Igmolecules, such as Protein A. For instance, a plurality ofinterferon-beta-1a/immunoglobulin fusion proteins may be bound toProtein A-agarose beads.

[0081] These polyvalent forms are useful since they possess multipleinterferon beta receptor binding sites. For example, a bivalent solubleinterferon-beta-1a may consist of two tandem repeats of amino acids 1 to166 of SEQ ID NO: 2 (or those encoded by nucleic acids numbered 1 to 498of SEQ. ID. NO:1) (moiety X in the generic formula) separated by alinker region (moiety Y), the repeats bound to at least a portion of animmunoglobulin constant domain (moiety Z). Alternate polyvalent formsmay also be constructed, for example, by chemically couplinginterferon-beta-1a/Ig fusions to any clinically acceptable carriermolecule, a polymer selected from the group consisting of Ficoll,polyethylene glycol or dextran using conventional coupling techniques.Alternatively, interferon-beta-1a may be chemically coupled to biotin,and the biotin-interferon beta Fc conjugate then allowed to bind toavidin, resulting in tetravalent avidin/biotin/interferon betamolecules. Interferon-beta-1a/Ig fusions may also be covalently coupledto dinitrophenol (DNP) or trinitrophenol (TNP) and the resultingconjugate precipitated with anti-DNP or anti-TNP-IgM, to form decamericconjugates with a valency of 10 for interferon beta receptor bindingsites.

[0082] The interferon-beta-1a proteins, fragments, and fusion proteinsof the invention may be isolated and purified in accordance withconventional conditions, such as extraction, precipitation,chromatography, affinity chromatography, electrophoresis or the like.For example, the interferon proteins and fragments may be purified bypassing a solution thereof through a column having an interferonreceptor immobilized thereon (see U.S. Pat. No. 4,725,669). The boundinterferon molecule may then be eluted by treatment with a chaotropicsalt or by elution with aqueous acetic acid. The immunoglobulin fusionproteins may be purified by passing a solution containing the fusionprotein through a column which contains immobilized protein A or proteinG which selectively binds the Fc portion of the fusion protein. See, forexample, Reis, K. J., et al., J. Immunol. 132:3098-3102 (1984); PCTApplication, Publication No. WO87/00329. The chimeric antibody may thenbe eluted by treatment with a chaotropic salt or by elution with aqueousacetic acid.

[0083] Alternatively the interferon proteins and immunoglobulin-fusionmolecules may be purified on anti-interferon antibody columns, or onanti-immunoglobulin antibody columns to give a substantially pureprotein. By the term “substantially pure” is intended that the proteinis free of the impurities that are naturally associated therewith.Substantial purity may be evidenced by a single band by electrophoresis.

[0084] An example of a useful interferon-beta-1a/Ig fusion protein ofthis invention is that of SEQ ID NO: 2, which is secreted into the cellculture by eukaryotic cells containing the expression plasmid pCMG261(See Example 2). This protein consists of the mature humaninterferon-beta-1a fused to a portion of the hinge region and the CH2and CH3 constant domains of murine Ig. This contains a sufficientportion of the murine immunoglobulin to be recognized by the Fc bindingprotein, Protein A.

[0085] Other fusion proteins of the invention incorporating humaninterferon-beta-1a are shown: (a) in SEQ ID NOS: 3 and 4 for the cDNAand deduced amino acids sequences, respectively of a histagged-interferon-beta-1a fusion (also shown in FIG. 1) and; (b) in SEQNO: 1 for the cDNA encoding the interferon-beta-1a/Ig fusion protein ofSEQ ID NO: 2 (also shown in FIG. 2).

[0086] The preferred interferon-beta-1a proteins of the inventioninclude the novel “junction” DNA sequence SEQ ID NO: 5 and amino acidSEQ ID NO: 6. SEQ ID NO: 5 represents the 11 triplet codons on eitherside of the junction between human interferon beta DNA and the DNAencoding a human immunoglobulin constant region (see Example 5: SEQ IDNOS: 41 and 42). Specifically, in SEQ ID NO: 5, the DNA encoding humaninterferon-beta-1a ends at nucleotide triplet 568-570 (AAC) and DNAencoding a human IgG1 constant region starts at the triplet (GAC)beginning with nucleotide number 574 of SEQ ID NO: 41. The correspondingdeduced amino acid “junction” sequence is represented in SEQ ID NO: 6and is based on SEQ ID NO: 42. Another unique “junction” sequence isdefined that includes a linker sequence in the final DNA construct (SeeExample 5: SEQ ID NOS: 43 and 44). This “junction” DNA and amino acidsequence are represented in SEQ ID NO: 7 and 8, respectively, whichshows the 11 triplet codons on either side of the junction directlybetween the end of the interferon-beta-1a sequence (nucleotide number570 in SEQ ID NO: 43) and the linker sequence (nucleotides 571 to 585 inSEQ ID NO:43; GGGGS in SEQ ID NO: 8).

[0087] The DNA “junction” sequences can be used as DNA probes and may bethe minimum DNA needed for hybridization under standard conditions toany DNA sequence encoding any interferon-beta-1a/Ig fusion protein.Nevertheless, provided that the whole probe hybridizes to both sides ofthe junction and both sides of the interferon beta/constant regionjunction participate in the hybridization, smaller sequences may exist.Furthermore, persons having ordinary skill in the art will understandthat DNA sequences larger than SEQ ID NO:5 or 7 will be suitable forhybridization as well. One of ordinary skill in the art can test if aparticular probe such as SEQ ID NO: 5 or 7 are capable of hybridizing onboth sides of the junction by labelling the 5′ end of either a singlestrand sense oligonucleotide or a single strand anti-senseoligonucleotide with an appropriately labelled phosphate of ATP usingpolynucleotide kinase. A sequence of the invention must hybridize to,and thus be labelled by both oligonucleotide probes. It is furtherunderstood that the invention encompasses fully degenerate sequencesencoding the junction SEQ ID NO: 5 or 7.

[0088] III. Other Variants of Interferon Fusion Polypeptides

[0089] Derivatives of proteins of the invention also include variousstructural forms of the primary protein which retain biologicalactivity. Due to the presence of ionizable amino and carboxyl groups,for example, interferon beta fusion protein may be in the form of acidicor basic salts, or may be in neutral form. Individual amino acidresidues may also be modified by oxidation or reduction. Further, theprimary amino acid structure (including the N- and/or C-terminal ends)or the glycan of the interferon-beta-1a may be modified (“derivatized”)by forming covalent or aggregative conjugates with other chemicalmoieties, such as glycosyl groups, polyalkylene glycol polymers such aspolyethylene glycol (PEG: see co-pending and commonly assignedapplication Serial Nos. 60/104,491 and 60/720,237), lipids, phosphate,acetyl groups and the like, or by creating amino acid sequence mutants.

[0090] Other derivatives of interferon beta/Ig include covalent oraggregative conjugates of interferon beta or its fragments with otherproteins or polypeptides, such as by synthesis in recombinant culture asadditional N-termini, or C-termini. For example, the conjugated peptidemay be a signal (or leader) polypeptide sequence at the N-terminalregion of the protein which co-translationally or post-translationallydirects transfer of the protein from its site of synthesis to its siteof function inside or outside of the cell membrane or wall (e.g., theyeast alpha-factor leader). Interferon beta receptor proteins cancomprise peptides added to facilitate purification or identification ofinterferon beta (e.g., histdine/interferon-beta-1a fusions). The aminoacid sequence of interferon beta can also be linked to the peptideAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (Hopp et al., Bio/Technology6:1204,1988.) The latter sequence is highly antigenic and provides anepitope reversibly bound by a specific monoclonal antibody, enablingrapid assay and facile purification of expressed recombinant protein.This sequence is also specifically cleaved by bovine mucosalenterokinase at the residue immediately following the Asp-Lys pairing.

[0091] Other analogs include interferon beta fusion Fc protein or itsbiologically active fragments whose interferon beta sequences differfrom those shown in SEQ ID NOS: 2, 4,6 or 8 by one or more conservativeamino acid substitutions or by one or more non conservative amino acidsubstitutions, or by deletions or insertions which do not abolish theisolated protein's biological activity. Conservative substitutionstypically include the substitution of one amino acid for another withsimilar characteristics such as substitutions within the followinggroups: valine, alanine and glycine; leucine and isoleucine; asparticacid and glutamic acid; asparagine and glutamine; serine and threonine;lysine and arginine; and phenylalanine and tyrosine. The non-polarhydrophobic amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Other conservativesubstitutions can be readily known by workers of ordinary skill. Forexample, for the amino acid alanine, a conservative substitution can betaken from any one of D-alanine, glycine, beta-alanine, L-cysteine andD-cysteine. For lysine, a replacement can be any one of D-lysine,arginine, D-arginine, homo-arginine, methionine, D-methionine,ornithine, or D-ornithine. Generally, substitutions that may be expectedto induce changes in the functional properties of isolated polypeptidesare those in which: (i) a polar residue, e.g., serine or threonine, issubstituted for (or by) a hydrophobic residue, e.g., leucine,isoleucine, phenylalanine, or alanine; (ii) a cysteine residue issubstituted for (or by) any other residue; (iii) a residue having anelectropositive side chain, e.g., lysine, arginine or histidine, issubstituted for (or by) a residue having an electronegative side chain,e.g., glutarnic acid or aspartic acid; or (iv) a residue having a bulkyside chain, e.g., phenylalanine, is substituted for (or by) one nothaving such a side chain, e.g., glycine. Included in the invention areisolated molecules that are: allelic variants, natural mutants, inducedmutants, proteins encoded by DNA that hybridize under high or lowstringency conditions to a nucleic acid which encodes a polypeptide suchas SEQ. ID. NOS.2, 4, 6 or 8.

[0092] We developed interferon-beta-1a mutants that are further variantsof the interferon-beta-1a moiety of the invention. Theseinterferon-beta-1a moieties may be particularly useful inasmuch as theydisplay novel properties not found in the wild type interferon-beta-1a(See Example 1). Briefly, we undertook a mutational analysis of humaninterferon-beta-1a with the aim of mapping residues required foractivity and receptor binding. The availability of the 3-D crystalstructure of human interferon-beta-1a (see Karpusas et al., 1997, Proc.Natl. Acad. Sci. 94: 11813-11818) allows us to identify, for alanine (orserine) substitutions, the solvent-exposed residues available forinterferon beta receptor interactions, and to retain amino acidsinvolved in intramolecular bonds. A panel of fifteen alanine scanningmutations were designed that replaced between two and eight residuesalong distinct regions each of the helices (A, B, C, D, E) and loops(AB1, AB2, AB3, CD1, CD2, DE1, DE2) of interferon-beta-1a. See Example1.

[0093] An amino-terminal histidine tag (“his” tag) was included foraffinity purification of mammalian cell expressed mutants (SEQ ID NO:2:FIG. 1). Functional consequences of these mutations were assessed inantiviral and antiproliferation assays. A non-radioactive binding assaywas developed to analyze these mutants for their binding to theinterferon beta surface cell receptor (IFNAR1/2 cell surface receptor).In addition, an ELISA-based assay employing an IFNAR2-ectodomain/Fcfusion protein to bind interferon was used to map interactions ofsurfaces between interferon-beta-1a and IFNAR2 (See Example 1). Thesemutational analyses demonstrated that N- and C-termini lie in a portionof the interferon-beta molecule not important for receptor binding orbiological function.

[0094] We have identified three types of effects that were caused bytargeted mutagenesis. These effects may be advantageous for interferondrug development under certain circumstances. The three types of effectare as follows: (a) mutants with higher antiviral activity than that ofhis-wild-type interferon-beta-1a (e.g. mutant C1); (b) mutants whichdisplay activity in both antiviral and antiproliferation assays, but forwhich antiproliferation activity is disproportionately low with respectto antiviral activity, compared to his-wild-type interferon-beta-1a(e.g., mutants C1, D and DE1); and (c) functional antagonists (e.g., A1,B2, CD2 and DE1), which show antiviral and antiproliferative activitiesthat are disproportionately low with respect to receptor binding,compared to his-wild-type interferon-beta-1a.

[0095] Moreover, the coupling between the interferon-beta-1a moiety (X)and the second, non-interferon-beta-1a moiety Z (e.g., an Fc region ofan immunoglobulin) can also be effected by any chemical reaction thatwill bind the two molecules together so long as the immunoglobulin andthe interferon-beta-1a retain their respective activities. This chemicallinkage can include many chemical mechanisms such as covalent binding,affinity binding, intercalation, coordinate binding and complexation.Representative coupling agents (i.e., linkers “Y” in the genericformula) to develop covalent binding between the interferon-beta-1a andimmunoglobulin moieties can include organic compounds such asthioesters, carbodiimides, succinimide esters, diisocyanates such astolylene-2,6-diisocyanate, gluteraldehydes, diazobenzenes andhexamethylene diamines such asbis-(p-diazonium-benzoyl)-ethylenediamine, bifunctional derivatives ofimidoesters such as dimethyl adipimidate, and bis-active fluorinecompounds such as 1,5-difluoro-2,4-dinitrobenzene. This listing is notintended to be exhaustive of the various classes of chemical couplingagents known in the art. Many of these are commercially available suchas N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC);4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene(SMPT: Pierce Chem. Co., Cat. # 21558G).

[0096] IV. Utility of the Invention

[0097] The fusion proteins of this invention can be used in therapeuticcompositions whenever interferon beta therapy is called for. Thesemolecules have the normal advantages associated with fusion proteins,particularly Ig fusions; namely an altered pharmacokinetics andpharmacodynamics, leading to increased half life and increased residencetime in the vasculature. Moreover, the particularly preferredglycosylated interferon-beta-1a proteins, although similar in structureto interferon beta 1b, are many times more biologically active than thenon-glycosylated interferon beta 1b. See Runkel et al., 1998, Pharm.Res. 15: 641-649.

[0098] The products of the present invention have been found useful insustaining the half life of therapeutic interferon-beta 1a, and may forexample be prepared for therapeutic administration by dissolving inwater or acceptable liquid medium. Administration is by either theparenteral, aerosol, or oral route. Fine colloidal suspensions may beprepared for parenteral administration to produce a depot effect, or bythe oral route while aerosol formulation may be liquid or dry powder innature. In the dry, lyophilized state or in solution formulations, theinterferon-beta-1a fusions of the present invention should have goodstorage stability.

[0099] The therapeutic proteins of the present invention may be utilizedfor the prophylaxis or treatment of any condition or disease state forwhich the interferon-beta-1a constituent is efficacious. In addition,the fusion proteins of the present invention may be utilized indiagnosis of constituents, conditions, or disease states in biologicalsystems or specimens, as well as for diagnosis purposes innon-physiological systems.

[0100] In therapeutic usage, the present invention contemplates a methodof treating an animal subject having or latently susceptible to suchcondition(s) or disease state(s) and in need of such treatment,comprising administering to such animal an effective amount of a fusionprotein of the present invention which is therapeutically effective forsaid condition or disease state. Subjects to be treated by the fusionsof the present invention include mammalian subjects and most preferablyhuman subjects. Depending on the specific condition or disease state tobe combated, animal subjects may be administered interferon-beta-1afusion proteins of the invention at any suitable therapeuticallyeffective and safe dosage, as may readily be determined within the skillof the art, and without undue experimentation. Because of the speciesbarriers of Type I interferons, it may be necessary to generateinterferon-fusion proteins as described herein with interferons from theappropriate species.

[0101] The anti-cell proliferative activity of interferon-beta-1a iswell known. In particular, certain of the interferon-beta-1a fusionsdescribed herein are useful for treating tumors and cancers such asosteogenic sarcoma, lymphoma, acute lymphocytic leukemia, breastcarcinoma, melanoma and nasopharyngeal carcinoma, as well as autoimmuneconditions such as fibrosis, lupus and multiple sclerosis. It is furtherexpected that the anti-viral activity exhibited by the fusion proteins,in particular certain of the interferon-beta-1a muteins describedherein, may be used in the treatment of viral diseases, such as ECMinfection, influenza, and other respiratory tract infections, rabies,and hepatitis. It is also expected that immunomodulatory activities ofinterferon-beta-1a exhibited by the proteins described herein, may beused in the treatment of autoimmune and inflammatory diseases, such asfibrosis, multiple sclerosis. The ability of interferons to inhibitformation of new blood vessels (angiogenesis or neovascularization)enable proteins of the invention to be used to treat angiogenic diseasessuch as diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma, retrolentalfibroplasia, rubeosis, and Osler-Webber Syndrome. Moreover, theantiendothelial activity of interferon has been known for some time andone potential mechanism of interferon action may be to interfere withendothelial cell activity by inhibiting the production or efficacy ofangiogenic factors produced by tumor cells. Some vascular tumors, suchas hemangiomas, are particularly sensitive to treatment with interferon.Treatment with interferon-alpha is the only documented treatment forthis disease. It is expected that treatment with the interferon-beta-1afusion proteins of the invention will offer subtantial pharmaceuticalbenefits in terms of pharmacokinetics and pharmacodynamics, since theconjugate is expected to remain in the vasculature for a longer periodof time than non-conjugated interferons, thus leading to more efficientand effective therapy for use as an anti-angiogenic agent. See Example9.

[0102] The polymer-interferon-beta-1a fusions of the invention may beadministered per se as well as in the form of pharmaceuticallyacceptable esters, salts, and other physiologically functionalderivatives thereof. In such pharmaceutical and medicament formulations,the interferon-beta-1a preferably is utilized together with one or morepharmaceutically acceptable carrier(s) and optionally any othertherapeutic ingredients. The carrier(s) must be pharmaceuticallyacceptable in the sense of being compatible with the other ingredientsof the formulation and not unduly deleterious to the recipient thereof.The interferon-beta-1a is provided in an amount effective to achieve thedesired pharmacological effect, as described above, and in a quantityappropriate to achieve the desired daily dose.

[0103] The formulations include those suitable for parenteral as well asnon-parenteral administration, and specific administration modalitiesinclude oral, rectal, buccal, topical, nasal, ophthalmic, subcutaneous,intramuscular, intravenous, transdermal, intrathecal, intra-articular,intra-arterial, sub-arachnoid, bronchial, lymphatic, vaginal, andintra-uterine administration. Formulations suitable for oral, nasal, andparenteral administration are preferred.

[0104] When the interferon-beta-1a is utilized in a formulationcomprising a liquid solution, the formulation advantageously may beadministered orally or parenterally. When the interferon-beta-1a isemployed in a liquid suspension formulation or as a powder in abiocompatible carrier formulation, the formulation may be advantageouslyadministered orally, rectally, or bronchially.

[0105] When the interferon-beta-1a is utilized directly in the form of apowdered solid, the interferon-beta-1a may advantageously beadministered orally. Alternatively, it may be administered nasally orbronchially, via nebulization of the powder in a carrier gas, to form agaseous dispersion of the powder which is inspired by the patient from abreathing circuit comprising a suitable nebulizer device.

[0106] The formulations comprising the proteins of the present inventionmay conveniently be presented in unit dosage forms and may be preparedby any of the methods well known in the art of pharmacy. Such methodsgenerally include the step of bringing the active ingredient(s) intoassociation with a carrier which constitutes one or more accessoryingredients. Typically, the formulations are prepared by uniformly andintimately bringing the active ingredient(s) into association with aliquid carrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into dosage forms of the desiredformulation.

[0107] Formulations of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets, tablets, or lozenges, each containing a predetermined amount ofthe active ingredient as a powder or granules; or a suspension in anaqueous liquor or a non-aqueous liquid, such as a syrup, an elixir, anemulsion, or a draught.

[0108] A tablet may be made by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, with the active compound being in afree-flowing form such as a powder or granules which optionally is mixedwith a binder, disintegrant, lubricant, inert diluent, surface activeagent, or discharging agent. Molded tablets comprised of a mixture ofthe powdered polymer conjugates with a suitable carrier may be made bymolding in a suitable machine.

[0109] A syrup may be made by adding the active compound to aconcentrated aqueous solution of a sugar, for example sucrose, to whichmay also be added any accessory ingredient(s). Such accessoryingredient(s) may include flavorings, suitable preservative, agents toretard crystallization of the sugar, and agents to increase thesolubility of any other ingredient, such as a polyhydroxy alcohol, forexample glycerol or sorbitol.

[0110] Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the active conjugate, whichpreferably is isotonic with the blood of the recipient (e.g.,physiological saline solution). Such formulations may include suspendingagents and thickening agents or other microparticulate systems which aredesigned to target the compound to blood components or one or moreorgans. The formulations may be presented in unit-dose or multi-doseform.

[0111] Nasal spray formulations comprise purified aqueous solutions ofthe active conjugate with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucus membranes.

[0112] Formulations for rectal administration may be presented as asuppository with a suitable carrier such as cocoa butter, hydrogenatedfats, or hydrogenated fatty carboxylic acid.

[0113] Ophthalmic formulations such as eye drops are prepared by asimilar method to the nasal spray, except that the pH and isotonicfactors are preferably adjusted to match that of the eye.

[0114] Topical formulations comprise the conjugates of the inventiondissolved or suspended in one or more media, such as mineral oil,petroleum, polyhydroxy alcohols, or other bases used for topicalpharmaceutical formulations.

[0115] In addition to the aforementioned ingredients, the formulationsof this invention may further include one or more accessoryingredient(s) selected from diluents, buffers, flavoring agents,disintegrants, surface active agents, thickeners, lubricants,preservatives (including antioxidants), and the like.

[0116] Accordingly, the present invention contemplates the provision ofsuitable fusion proteins for in vitro stabilization of interferon-beta1a in solution, as a preferred illustrative application ofnon-therapeutic application. The fusion proteins may be employed forexample to increase the resistance to enzymatic degradation of theinterferon-beta 1a. and provides a means of improving shelf life, roomtemperature stability, and robustness of research reagents and kits.

[0117] The following Examples are provided to illustrate the presentinvention, and should not be construed as limiting thereof. Inparticular, it will be understood that the in vivo, animal experimentsdescribed herein may be varied, so that other modifications andvariations of the basic methodology are possible. For example, inExample 7, one of ordinary skill in the art could use other neopterinassays or could alter the number and kind of primate used. Thesemodifications and variations to the Examples are to be regarded as beingwithin the spirit and scope of the invention.

EXAMPLE 1 Structure/Activity Studies of Human Interferon-beta-1a UsingAlanine/Serine Substitution Mutations: Analysis of Receptor BindingSites and Functional Domains

[0118] A. Overview

[0119] An extensive mutational analysis of human interferon-beta-1a(IFN-beta-1a) was undertaken with the aims of mapping residues requiredfor activity and receptor binding. The availability of the 3-D crystalstructure of human IFN-beta (Karpusas, M. et al. 1997, Proc. Natl. Acad.Sci. 94: 11813-11818) allowed us to identify for alanine (or serine)substitutions the solvent-exposed residues available for receptorinteractions, and to retain amino acids involved in intramolecularbonds. A panel of 15 alanine substitution mutations were designed thatreplaced between 2 and 8 residues along distinct regions of each of thehelices (A, B, C, D, E) and loops (AB, CD, DE). An amino-terminal tagconsisting of 6 histidine residues was included for affinitypurification, as well as an enterokinase linker sequence site forremoval of the amino-terminal extension. The resulting interferons areinterchangeably referred to as “his tagged-interferon(IFN)-beta” orHis₆-interferon-beta” and the like.

[0120] Various mutant his tagged-IFN-beta expression plasmids wereconstructed using a wild type IFN-beta gene construct as a template formutagenesis. The mutagenesis strategy involved first introducing uniquerestriction enzyme cleavage sites throughout the wild type histagged-IFN beta gene, then replacing distinct DNA sequences between thechosen restriction sites with synthetic oligonucleotide duplexes, whichencoded the alanine (or serine) substitution mutations. Finally, themutant IFN genes were subcloned into a plasmid which directed mammaliancell expression in a human 293 kidney cell line.

[0121] Functional consequences of these mutations were assessed inantiviral and antiproliferation assays. A non-radioactive IFN bindingassay was developed to analyze these mutants in their binding to thesurface receptor (“IFNAR1/2 complex”) of human Daudi Burkitt's lymphomacells. In addition, an assay to map interaction surfaces betweenhis-IFN-beta mutants and IFNAR2 was developed that employed a IFNAR2/Fcfusion protein, comprised of the IFN receptor protein IFNAR2extracellular domain fused to the hinge, CH2 and CH3 domains of humanIgG1.

[0122] 1. Creation of an Interferon Beta Gene as a Template forMutagenesis

[0123] Our strategy to generate IFN-beta alanine (or serine) substitutedmutants was to first create a modified IFN-beta gene, which encoded thewild type protein but which carried unique restriction enzyme cleavagesites scattered across the gene. The unique sites were used to exchangewild type sequences for synthetic oligonucleotide duplexes, which encodethe mutated codons. In order to obtain an human IFN-beta-1a expressioncassette suitable for creation of mutant genes, the IFN-beta cDNA(GenBank accession #E00029) was amplified by PCR. An initial cloning ofthe IFN-beta gene into plasmid pMJB107, a derivative of pACYC184, seeRose, et. al., 1988, Nucleic Acids Res. 16 (1)355) was necessary inorder to perform site-directed mutagenesis of the gene in a plasmid thatlacked the specific restriction sites which would be generated throughthe mutagenesis.

[0124] The PCR primers used to subclone the coding sequences of thehuman IFN-beta gene also allowed us to introduce an enterokinase linkersequence upstream and in frame with the IFN-beta gene (5′ PCR primer5′TTCTCCGGAGACGATGATGACAAGATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGC-3′ (SEQID NO: 9: “BET-021”, and 3′ PCR primer5′-GCCGCTCGAGTTATCAGTTTCGGAGGTAACCTGTAAGTC-3′ (SEQ ID NO:10: “BET-022”)and flanking restriction enzyme sites (BspEI and Xho I) useful forcloning into plasmid pMJB107 sites. The resulting DNA is refererred toas PCR fragment A.

[0125] An efficient signal sequence from the human vascular celladhesion molecule-I (VCAM-1) signal sequence and a six histidine tagwere introduced into the final construct from a second DNA fragmentcreated from pDSW247 (fragment B). Plasmid pDSW247 is a derivative ofpCEP4 (Invitrogen, Carlsbad, Calif.) from which the EBNA-1 gene has beendeleted, and which carries the VCAM-1 signal sequence (VCAMs) fusedupstream and in frame with a six histidine tag. The PCR primers thatwere used to generate the VCAMss-1/histidine tag cassette moiety wereKID-369 (5′ PCR primer 5′-AGCTTCCGGGGGCCATCATCATCATCATCATAGCT-3′: SEQ IDNO: 11) and KID-421 (3′ PCR primer 5′-CCGGAGCTATGATGATGATGATGATGGCCCCCGGA-3′: SEQ ID NO: 12) incorporating flanking restriction enzymecleavage sites (NotI and BspEI) that allowed excision of the fragment BDNA.

[0126] To create a plasmid vector that carried the VCAM-1 signalsequence, his tag and interferon-beta gene we performed a three-wayligation using gel purified DNA fragments from plasmid vector pMJB107(NotI and XhoI cleaved), PCR fragment A (BspEI and XhoI cleaved) andfragment B (NotI and BspEI cleaved). The ligated plasmid was used totransform either JA221 or XL1-Blue E. coli cells and ampicillinresistant colonies were picked and tested for inserts by restriction mapanalysis. Maxiprep DNA was made and the sequence of the insert wasverified by DNA sequencing. The resulting construct was called pCMG260.

[0127] 2. Creation of Alanine Substitution Mutants of HumanInterferon-beta in pCMG260

[0128] The plasmid pCMG260 was used as a template for multiple rounds ofmutagenesis (U.S.E. Site Directed Mutagenesis Kit (Boehringer-Mannheim),which introduced unique restriction cleavage sites into positions alongthe IFN-beta protein coding sequence but did not change the resultingsequence of the protein. The mutagenized plasmids were used to transformeither the JA221 or XL1-Blue strains of E. coli and recombinant coloniesselected for chloramphenicol resistance. Chloramphenicol resistantcolonies were further tested for the presence of the desired uniquerestriction enzyme site by DNA restriction mapping analysis. Theresulting IFN-beta plasmid, pCMG275.8, contained the full set of uniquerestriction enzyme cleavage sites and the DNA sequence of the gene wasverified. The full DNA sequence of the modified, his-tagged interferonbeta gene, together with the wild type protein coding sequence, aregiven in FIG. 1.

[0129] The full set of alanine substitution mutations are depicted inTable 1 (next page). The names of the mutants specify the structuralregions (helices and loops) in which the mutations were introduced. Theentire panel of alanine (serine) substitutions results in mutation of 65of the 165 amino acids of human IFN-beta.

[0130] The panel of mutants was created from pCMG275.8 by replacingsegments of DNA between the unique restriction sites with syntheticoligonucleotide duplexes, which carried the genetic coding informationdepicted in Table 2 (see below). To create the various alaninesubstitution mutant plasmids, gel purified pCMG275.8 vector (cleavedwith the appropriate restriction enzyme, as indicated on the list belowfor each IFN-beta structural region) and oligonucleotide duplexes(coding strand sequences are shown in Table 2) were ligated together.The ligation mixtures were used to transform the JA221 strain of E. coliand recombinant colonies selected for ampicillin resistance. Ampicillinresistant colonies were tested for the presence of the insertion of themutations by screening for appropriate restriction enzyme sites. For twomutants (A2 and CD2), the cloning strategy entailed using two duplexesof synthetic oligonucleotides (shown in Table 2), which carrycomplementary overhanging ends to allow them to ligate to each other andwith the vector-IFN-beta backbone in a three-way ligation. The followinglist illustrates the sites which were used to clone the mutatedoligonucleotides from Table 2. The cloning scheme (subsection B) showsthe positions of these unique sites on the interferon beta gene. A helixBspEI to MunI or BgIII to Pst I AB loop MunI to PstI or MunI to BsaHI Bhelix BspHI to BsaI or BsaHI to BsaI C helix BsaI to XbaI CD loop XbaIto BspHI or XbaI to DraIII D helix BspHI to DraIII DE loop BspHI to PvuIE helix PvuI to BstEII

[0131] TABLE 1 Positions of alanine substitution mutations of ^(HU)IFN-β1        10        20        30        40        50 |. .. . |. .. ..| ....|. .. .. .| . .....|... IFN-βMSYNLLGFLQRSSNFQCQKLLWQWGPLEYCLKDRMNFDIPEEIKQLQQFQKE A1-A-AA--A--A----------------------------------------- A2--------------AA-AA--AA----------------------------- AB1---------------------------AAA-AA------------------- AB2-----------------------------------AA-A--A---------- AB3--------------------------------------------AAAAA-AA _(|———————)helixA_(————————| |————————)AB loop_(———————————|)       60        70        80        90        100       |    . ..| ... | .. .. .|  . . .|... ... IFN-β DAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKE B1----------A--AS----------------------------------------- B2-----------------AAA------------------------------------ C1---------------------------AS--AA--S-------------------- C2--------------------------------------A---A--AA--------- CD1-------------------------------------------------AA--AAA_(|————————)helix B_(——| |————————————————————————| |—)CD loop_(——) 110     120        130 140       150        160  |.. . . .|        .|.. ... .|. . |  . . . | IFN-βDFTRGALMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYRINRLTGYLRN CD2AA-A--A--A----------------------------------------------- D------------------A-AA--A-------------------------------- DE1-------------------------AA------------------------------ DE2---------------------------AA---------------------------- E------------------------------------A---A--A--A---------- CDloop_(—| |————)helix D_(————|    |———————)helix E_(——————————|)

[0132] The line designated IFN-β shows the wild type human IFN-βsequence. Alanine or serine substitutions of the IFN-β residues areshown for each of the mutants and dashes, below relevant regions,indicate wild type sequences. The helices and loop structures areindicated as solid lines below the mutants. The DE loop spans the gapbetween the D and E helices. Two additional alanine substitution mutants(H93A, H97A and H121A) were generated and analyzed in antiviral assaysto assess the effects of mutating these histidines, which chelate zincin the crustal structure dimer. Both of these mutants retained full wildtype activity in antiviral assays, suggesting that zinc-mediated dimerformation is not important for IFN-β activity. TABLE 2 A1 SEQ IDCCGGAGACGATGATGACAAGATGGCTTACGCCGCTCTTGGAGCCCTACAAG NO: 13CTTCTAGCAATTTTCAGTGTCAGAAGCTCCTGTGGC BET-053 A2 SEQ IDGATCTAGCAATGCTGCCTGTGCTGCCCTCCTGGCTGCCTTGAATGGGAGGC NO: 14 TTGAATACTBET-039 SEQ ID GCCTCAAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCA NO:15 BET-041 AB1 SEQ IDAATTGAATGGGAGGGCTGCAGCTTGCGCTGCAGACAGGATGAACTTTGACAT NO: 16CCCTGAGGAGATTAAGCAGCTGCA BET-080 AB2 SEQ IDAATTGAATCGGAGGCTTGAATACTGCCTCAAGGACAGGGCTGCATTTGCTAT NO: 17CCCTGCAGAOATTAAGCAGCTGCA BET-082 AB3 SEQ IDAATTGAATGGGAGGCTTGAATACTGCCTCAAGGACAGGATGAACTTTGACA NO: 18 BET-084 SEQID TCCCTGAGGAGATTGCTCCAGCTOCAGCTTTCGCTGCAGCTGA NO: 19 BET-086 B1 SEQ IDCGCCGCGTTGACCATCTATGAGATGCTCGCTAACATCGCTAGCATTTTCAGA NO: 20CAAGATTCATCTACCACTGGCTGGAA BET-110 B2 SEQ IDCGCCGCATTGACCATCTATGAGATGCTCCAGAACATCTTTGCTATTTTCGCT NO: 21GCAGCTTCATCTAGCACTGGCTGGAA BET-112 C1 SEQ IDGGAATGCTTCAATTGTTGCTGCACTCCTGAGCAATGTCTATCATCAGATAAA NO: 22CCATCTGAAGACAGTTCTAG BET-114 C2 SEQ IDGGAATGAGACCATTGTTGAGAACCTCCTGGCTAATGTCGCTCATCAGATAGC NO :23ACATCTGGCTGCAGTTCTAG BET-092 CD1 SEQ IDCTAGCTGCAAAACTGGCTGCAGCTGATTTCACCAGGGGAAAACT NO: 24 BET-094 CD2 SEQ IDCTAGAAGAAAAACTGGAGAAACAAGCAGCTACCGCTGCAAAAGCAATGAGCG NO: 25CGCTGCACCTGAAAAGA BET-096 SEQ IDTATTATGGGAGGATTCTCCATTACCTGAAGGCCAAGGAGTACTCACACTGT NO: 26 BET-106 D1SEQ ID CATGAGCAGTCTGCACCTGAAAAGATATTATGGOGCAATTGCTGCATACCT NO: 27CGCAGCCAAGGAGTACTCACACTGT BET-108 DE1 SEQ IDCATGAGCAGTCTGCACCTCAAAAGATATTATGGGAGGATTCTGCATTACCT NO: 28GAAGGCCGCTGCATACTCACACTGTGCCTGGACGAT BET-116 DE2 SEQ IDCATGAGCAGTCTGCACCTGAAAAGATATTATGGGAGGATTCTGCATTACCTG NO: 29AAGGCAAAGGAGTACGCTGCATCTGCCTGGACGAT BET-118 E1 SEQ IDCGTCAGAGCTGAAATCCTAGCAAACTTTGCATTCATTGCAAGACTTACAG NO: 30 BET-104

[0133] B. Construction of EBNA 293 expression plasmids

[0134] The wild type and mutant IFN-beta genes, fused to the VCAM-1signal sequence, his tag and enterokinase linker sequence, were gelpurified as 761 base pair NotI and BamHI restriction fragments. Thepurified genes were subcloned into NotI and BamHI cleaved plasmid vectorpDSW247, which is a derivative of pCEP4 (Invitrogen, Carlsbad, Calif.).Plasmid pDSW247 is an expression vector for transient expression ofprotein in human EBNA 293 kidney cells (Invitrogen, Carlsbad, Calif.).It contains the cytomegalovirus early gene promoter and EBV regulatoryelements which are required for high level gene expression in thatsystem, as well as selectable markers for E. coli (ampicillinresistance) and EBNA 293 cells (hygromycin resistance). The ligatedplasmids were used to transform either JA221 or XL1-Blue E. coli cellsand ampicillin resistant colonies were picked and tested for inserts byrestriction map analysis. Maxiprep DNA was made and the sequence of theinserts was verified by DNA sequencing. Positive clones displaying thedesired mutagenized sequences were used to transfect human EBNA 293kidney cells.

[0135] The overall cloning and expression strategy is presented in FIG.12.

[0136] C. Expression and Quantitation of IFN-beta-1a AlanineSubstitution Mutants

[0137] The human EBNA 293 cells (Invitrogen, Carlsbad, Calif.,Chittenden, T. (1989) J. Virol. 63: 3016-3025) were maintained assubconfluent cultures in Dulbecco's Modified Eagle's media supplementedwith 10% fetal bovine serum, 2 mM glutamine and 250 μg/ml Geneticin(Life Technologies, Gaithersburg, Md.). The pDSW247 expression plasmidswere transiently transfected into EBNA 293 cells using the lipofectamineprotocol (Gibco/BRL, Life Technologies). Conditioned media was harvested3-4 days posttransfection, cell debris was removed by centrifugation,and the his-IFN-beta concentration was quantitated by ELISA.

[0138] The ELISA assay was performed using polyclonal rabbit antibodies(protein A purified IgG, antibodies had been raised to purified humanIFN-beta-1a) to coat 96-well ELISA plates and a biotinylated form of thesame polyclonal rabbit Ig was used as a secondary reagent to allowinterferon detection using streptavidin-linked horseradish peroxidase(HRP: Jackson ImmunoResearch, W. Grove, Pa.). A dilution series ofinterferon-beta-1a (as AVONEX® sold by Biogen, Inc.) was used togenerate standard concentration curves. The his-IFN-beta containingconditioned media from the EBNA transfectants were diluted to obtainsamples with concentrations ranging between 10 g/ml and 0.3 ng/ml in theELISA assay. To confirm the concentrations of the IFN-beta in mediadetermined by ELISA, western blot analysis was performed. Reducedculture supernatants and IFN-beta-1a standards were subjected toSDS-PAGE on 10-20% gradient gels (Novex, San Diego, Calif.) and blottedonto PDVF membranes. Immunoreactive bands were detected with a rabbitpolyclonal anti-IFN-beta-1a antiserum (#447, Biogen, Inc., a secondantiserum that had been raised against IFN-beta-1a), followed bytreatment with HRP-linked donkey anti-rabbit IgG (JacksonImmunoResearch, W. Grove, Pa.).

[0139] D. Assessing the Interferon-Beta Mutants for Receptor Binding

[0140] The receptor binding properties of the Interferon-beta mutantsdescribed in C were assessed using two different binding assays. Oneassay measured binding of the interferon-beta mutants to a fusionprotein, IFNAR2/Fc, comprising the extracellular domain of the humanIFNAR2 receptor chain fused to part of the constant region of a humanIgG. IFNAR2-Fc was expressed in chinese hamster ovary (CHO) cells andpurified by protein A sepharose affinity chromatography according to theinstructions of the manufacturer (Pierce Chem. Co., Rockford, Ill.,catalog #20334). The binding of interferon-beta mutants to IFNAR2-Fc wasmeasured in an ELISA format assay. ELISA plates were prepared by coatingflat-bottomed 96 well plates overnight at 4° C. with 50 μl/well of mouseanti-human IgG1 monoclonal antibody (CDG5-AA9, Biogen, Inc.) at 10 μg/mlin coating buffer (50 mM NaHCO₃, 0.2 mM MgCl₂, 0.2 mM CaCl₂, pH 9.6).Plates were washed twice with PBS containing 0.05% Tween-20, and blockedwith 0.5% non-fat dry milk in PBS for 1 hour at room temperature. Aftertwo more washes, 50 μl of 1 μg/ml IFNAR2-Fc in 0.5% milk in PBScontaining 0.05% Tween-20 was added to each well and incubated for 1hour at room temperature, and the plates were then washed twice more.Binding of the interferon-beta mutants to IFNAR2-Fc was measured byadding 50 μl/well mutant interferon-beta in conditioned media, seriallydiluted in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with10% fetal bovine serum, and incubating for 2 hours at 4° C. Dilutions ofinterferon-beta mutant typically ranged from approximately 1 μM down to10 pM. After washing, interferon-beta bound to the plates was detectedby adding 50 μl/well of a cocktail consisting of a 1:1000 dilution of arabbit polyclonal anti-interferon antibody (#447, Biogen, Inc.) plushorseradish peroxidase (HRP)-labelled donkey anti-rabbit IgG (JacksonImmunoResearch), and incubating for 15 minutes at 4° C. After twowashes, HRP substrate was added, and the plate was incubated at 4° C.before being read on an ELISA plate reader at an absorbance of 450 nm.Data were plotted as absorbance versus the concentration of mutantinterferon-beta, and the affinity for the binding of the mutantinterferon-beta to IFNAR2-Fc was determined by fitting the data to asimple hyperbolic binding equation. Results from these analyses areshown in FIG. 3, in which the binding affinity for each mutant,determined in triplicate experiments, is expressed as a percentage ofthat measured for His₆-wild-type interferon-beta-1a.

[0141] A second receptor binding assay was used to measure the affinitywith which the interferon-beta mutants bound to Daudi cells expressingboth receptor chains, IFNAR1 and IFNAR2, which together comprise thereceptor for interferon-beta. This FACS-based assay used a blockingmonoclonal antibody directed against the extracellular domain of IFNAR1,EA12 (Biogen, Inc.), to distinguish unoccupied (free) receptor fromreceptor to which interferon-beta was bound. Daudi cells (20 μl at2.5×10⁷ cells/ml) were placed in 96-well V-bottom ELISA plates, andincubated for 1 hour at 4° C. with various concentrations ofinterferon-beta mutant (20 μl in FACS buffer; 5% FBS, 0.1% NaN₃ in PBS).Desirable serial dilutions of interferon-beta mutants ranged from 0.5 μMdown to 0.5 pM. To each well was added 100 ng of biotinylated murineanti-IFNAR1 monoclonal antibody EA12 (10 μl), and the plates incubatedfor an additional 2 minutes at room temperature before being washedtwice with FACS buffer (4° C.). The cells were then incubated for 30minutes at 4° C. with 50 μl/well of a 1:200 dilution ofR-Phycoerythrin-conjugated streptavidin (Jackson ImmunoResearch, WestGrove, Pa.), washed twice in FACS buffer, resuspended in 300 μl FACSbuffer containing 0.5% paraformaldehyde, and transferred into 12×75 mmpolystyrene tubes (Falcon 2052). The samples were then analyzed by flowcytometry on a FACScan (Becton Dickinson). Data were plotted as meanchannel fluorescence intensity (MFCI) versus the concentration ofinterferon-beta mutant; binding affinities were defined as theconcentration of interferon-beta mutant giving 50% inhibition ofantibody staining. Each mutant was tested multiple times. FIG. 4 showsthe receptor binding affinities for each interferon-beta mutant,determined by this method, expressed as a percentage of the affinitymeasured for His₆-wild-type interferon-beta-1a in each experiment.

[0142] E. Assessing the Interferon-Beta Mutants for Function

[0143] The interferon-beta mutants were also tested for functionalactivity using in vitro assays for antiviral activity and for theability of the interferon-beta to inhibit cell proliferation. A minimumof three antiviral assays, each with triplicate data points, wereperformed on each mutant. His₆-wild-type interferon-beta-1a was includedas a reference in every experiment. The antiviral assays were performedby treating A549 human lung carcinoma cells (ATCC CCL 185) overnightwith 2-fold serial dilutions of mutant interferon-beta at concentrationsthat spanned the range between full antiviral protection and noprotection from viral cell killing. The following day, the cells werechallenged for two days with encephalomyocarditis virus (ECMV) at adilution that resulted in complete cell killing in the absence ofinterferon. Plates were then developed with the metabolic dye MTT(2,3-bis[2-Methoxy-4-nitro-5-sulfo-phenyl]-2H-tetrazolium-5-carboxyanilide)(M-5655, Sigma, St. Louis, Mo.). A stock solution of MTT was prepared at5 mg/ml in PBS and sterile filtered, and 50 μl of this solution wasdiluted into cell cultures (100 μl per well). Following incubation atroom temperature for 30-60 minutes, the MTT/media solution wasdiscarded, cells were washed with 100 μl PBS, and finally themetabolized dye was solubilized in 100 μl 1.2N hydrochloric acid in 90%isopropanol. Viable cells (as evidenced by the presence of the dye) werequantified by absorbance at 450 nm. Data were analyzed by plottingabsorbance against the concentration interferon-beta mutant, and theactivity of each mutant was defined as the concentration at which 50% ofthe cells were killed. FIG. 5 shows the activity of each mutantexpressed as a percentage of the activity measured for histagged-wild-type interferon-beta-1a in each experiment.

[0144] Interferon-beta mutants were also assessed for function in anantiproliferation assay. Human Daudi Burkitt's lymphoma cells (ATCC #CCL 213) were seeded at 2×10⁵ cells/ml in RPMI 1620 supplemented with10% defined fetal calf serum (Hyclone, Logan Utah), and 2 mML-glutamine. Each well also contained a given concentration ofinterferon-beta mutant in a final total volume of 100 μl of medium perwell; the interferon-beta concentrations used were chosen to span therange from maximal inhibition of Daudi cell proliferation to noinhibition (i.e. full proliferation). Duplicate experimental points wereused for each concentration of interferon-beta mutant tested, and aduplicate set of untreated cells was included in all experiments. Cellswere incubated for two days at 37° C. in 5% CO₂ incubators, after which1 μCi per well of tritiated thymidine ((methyl-³H) thymidine, AmershamTRK758) in 50 μl medium was added to each well, and incubated for afurther 4 h. Cells were harvested using a LKB plate harvester, andincorporation of tritiated thymidine was measured using a LKB beta platereader. Duplicate experimental values were averaged and the standarddeviations determined. Data were plotted as mean counts per minuteversus the concentration of interferon-beta mutant, and the activity ofeach mutant was defined as the concentration required to give 50% of themaximal observed growth inhibition. Multiple assays for each mutant wereperformed. FIG. 6 shows the results expressed as a percentage of theactivity found for his tagged-wild-type interferon-beta-1a in eachexperiment.

[0145] F. Properties of the Interferon-Beta Mutants

[0146] Histidine tagged-wild-type interferon-beta-1a was found to haveactivities in the antiviral and antiproliferation assays that were eachabout 3-fold lower than the corresponding activities found for untaggedwild-type interferon-beta-1a. Because all of the interferon-beta mutantsA1-E contain the identical his tag sequence at their N-termini, theeffects of the mutations on the properties of the molecule weredetermined by comparing the activities of these mutants in theantiviral, antiproliferation and binding assays to the activity observedfor his tagged-wild-type interferon-beta-1a. In so doing, we assume thatvariations in the activities of mutants A1-E, compared to histagged-wild-type interferon-beta-1a, are qualitatively andquantitatively about the same as the effects that these same mutationswould have in the absence of the N-terminal his tag. The equivalentassumption for tagged or fusion constructs of other soluble cytokines iscommonly held to be true by practitioners of the technique of alaninescanning mutagenesis, especially when the in vitro functional activityof the tagged or fusion construct is close to that of the wild-typecytokine as is the case here. See, for example, Pearce K. H. Jr, et al.,J. Biol. Chem. 272:20595-20602 (1997) and Jones J. T., et al., J. Biol.Chem. 273:11667-11674 (1998)

[0147] The data shown in FIGS. 3-6 suggests three types of effects thatwere caused by the targeted mutagenesis. These effects may beadvantageous for interferon drug development under certaincircumstances. The three types of effect are as follows: (a) mutantswith higher antiviral activity than that of wild-type interferon-beta-1a(e.g. mutant C1); (b) mutants which display activity in both antiviraland antiproliferation assays, but for which antiproliferation activityis disproportionately low with respect to antiviral activity, comparedto wild-type interferon-beta-1a (e.g., mutants C1, D and DE1); and (c)functional antagonists (e.g., A1, B2, CD2 and DE1), which show antiviraland antiproliferative activities that are disproportionately low withrespect to receptor binding, compared to wild-type interferon-beta-1a.It can be seen that some mutants fall into more than one class. Theseclasses are reviewed below. While we have characterized these classes ofmutants with respect to those examples listed, it should be appreciatedthat other mutations in these regions may result in similar, or evenenhanced effects on activity:

[0148] a) Mutant C1 possesses antiviral activity that is approximately6-fold greater than that of wild-type his-tagged interferon-beta-1a.This mutant and others of this type are predicted to be useful inreducing the amount of interferon-beta that must be administered toachieve a given level of antiviral effect. Lowering the amount ofadministered protein is expected to reduce the immunogenicity of theprotein and may also reduce side-effects from non-mechanism-basedtoxicities. Mutations in this class are predicted to be advantageous insituations where the therapeutic benefit of interferon-betaadministration results from its antiviral effects, and whereantiproliferative effects contribute to toxicity or to unwantedside-effects.

[0149] (b) The relative activities (% wild type) of the alaninesubstituted mutants in antiviral and antiproliferation assay arecompared in FIG. 7. Coordinately changed activities (i.e. antiviral andantiproliferation activities that differ by the same factor from theactivities of the wild-type his tagged-interferon-beta-1a) are seen inmost mutants (those lying on the diagonal line). However, severalmutants show greater alterations in activity in one assay relative tothe other, compared to wild-type his tagged-interferon-beta-1a, asevidenced by displacement from the diagonal. Three such mutants areshown in the Table below. Mutant C1 shows antiviral activity that is˜6-fold higher than that of wild-type his tagged-interferon-beta-1a, butits activity in the antiproliferation assay is similar to that ofwild-type. Mutant C1 thus has antiviral activity that is enhanced by afactor of 5.2 over its antiproliferation activity, relative to wild-typehis tagged-interferon-beta-1a. Similarly, mutant D displays 65% of wildtype activity in the antiviral assay, but only 20% of wild-type activityin the antiproliferation assay, and thus has antiviral activity that isenhanced 3.4-fold over its antiproliferation activity compared to wildtype. Mutant DE1 displays 26% of wild type activity in the antiviralassay but only 8.5% in the antiproliferation assay, and thus hasantiviral activity that is enhanced 3.0-fold over its antiproliferationactivity compared to wild-type his tagged-interferon-beta-1a. Whenadministered at a concentration sufficient to achieve a desired level ofantiviral activity, these mutant proteins will show substantially lowerlevels of antiproliferative activity than the wild-type protein.Mutations in this class, like those in class (a), are predicted to beadvantageous in situations where the therapeutic benefit ofinterferon-beta administration results from its antiviral effects, andwhere antiproliferative effects contribute to toxicity or to unwantedside-effects. Antiviral Activity Antiproliferative (AP) (AV) Activity (%wild Mutant (% wild type) type) AV/AP C1 571 109 5.2 D 65 19 3.4 DE1 268.5 3.0

[0150] (c) Mutants with antiviral and antiproliferative activities thatare low with respect to receptor binding, as compared to wild-type histagged-interferon-beta-1a (see Table below). Mutant A1 displaysantiviral and antiproliferative activities that are 2.0-fold and1.8-fold higher than that observed for wild-type histagged-interferon-beta-1a, but binds to the cognate receptor on Daudicells with an affinity that is 29-fold higher than wild-type. Thebinding of this mutant to the IFN-beta receptor is thus enhancedapproximately 15-fold compared to the antiviral and antproliferationactivities of the protein. Similarly, mutants B2, CD2 and DE1 showenhancements of binding over antiviral activity of 4.6-, 4.6- and18-fold, respectively, and over antiproliferation activity of 3.5-, 15-and 54-fold. These proteins are predicted to be useful as functionalantagonists of the activity of endogenous IFN-beta, and possibly ofother endogenous Type I interferons, because they have the ability tobind to and occupy the receptor, and yet generate only a small fractionof the functional response in the target cells that would be seen withwild type IFN-beta. Cell Antiviral Antiproliferat- Binding Activity iveActivity Activity Binding Mutant (AV) (% wt) (AP) (% wt) (% wt)Binding/AV /AP A1 200 180 2900 15 16 B2 7.1 9.2 33 4.6 3.5 CD2 150 46690 4.6 15 DE1 26 8.5 460 18 54

[0151] G. Mutein Relationship to Three Dimensional Structure ofInterferon

[0152] While published crystal structures for a non-glycosylated form ofmurine interferon beta (T. Senda, S. Saitoh and Y. Mitsui. RefinedCrystal Structure of Recombinant Murine Interferon-β at 2.15 ÅResolution. J. Mol. Biol. 253: 187-207 (1995)) and for human interferonalpha-2b (R. Radhakrishnan, L. J. Walter, A. Hruza, P. Reichert, P. PTrotta, T. L. Nagabhushan and M. R. Walter. Zinc Mediated Dimer of HumanInterferon-α2b Revealed by X-ray Crystallography. Structure. 4:1453-1463 (1996)) had provided models for the polypeptide backbone ofhuman interferon beta, we have recently solved the structure forinterferon-beta-1a in its glycosylated state (M. Karpusas, M. Nolte, C.B. Benton, W. Meier, W. N. Lipscomb, and S. E Goelz. The CrystalStructure of Human Interferon-β at 2.2 Å resolution. Proc. Natl. Acad.Sci. USA 94: 11813-11818 (1997)).

[0153] The results of our mutational analyses can be summarized withrespect to the 3D-structure of interferon-beta-1a (not presented here).Certain mutatations residues created a reduction in activity (2 to >5fold reduced). The mutated regions correspond to the substitutions givenin Tables 1 and 2.

[0154] Mutations that are most significant in their effect on functionresulted in a dramatic reduction in both activity and cell surfacereceptor binding. Mutations in this region (A2 helix, AB& AB2 loop and Ehelix) correspond to mutations in the IFNAR2 binding site, since none ofthese mutants bound IFNAR/Fc in our assay.

[0155] While those mutations that were important for IFNAR2 binding alsoaffected cell binding, cell surface binding properties are alsoinfluenced by residues in other regions of the molecule (B1 helix, C2helix). It can be seen in the 3-D models (not shown) depicting theeffects of the alanine substitution mutants that the N-terminal,C-terminal and the glycosylated C helix regions of the IFN-beta-1amolecule do not lie within the receptor binding site. Mutations in theseregions did not reduce biological activity or reduce cell surfacereceptor binding.

EXAMPLE 2 Construction of Plasmids for Expression of Interferon-beta-1aFusion (IFN-beta/Fc) Protein

[0156] PCR technology was employed to create an expression plasmidencoding the human IFN-beta DNA sequence fused to the Fc portion ofmurine IgG2a heavy chain molecule. The plasmid vector pDSW247 (seeExample 1) is a derivative of pCEP4 (Invitrogen, Carlsbad, Calif.), fromwhich the EBNA-1 gene has been deleted. This plasmid was used for theconstruction of an expression vector useful for transient proteinexpression in EBNA 293 human kidney cells (Invitrogen, Carlsbad, Calif.,Shen. E. S., et. al. 1995, Gene 156, 235-239). It was designed tocontain a human vascular cell adhesion molecule-I (VCAM-1) signalsequence in frame and upstream of the interferon beta sequence, and anenterokinase linker sequence at the junction of the interferon beta andIg sequences.

[0157] The fusion protein expression cassette was assembled from severalDNA fragments. To obtain a DNA fragment encoding the human IFN-betagene, the cDNA subclone of human IFN-beta (GenBank accession #E00029)was used as a template for PCR using primers(5′-GGTGGTCTCACATGAGCTACAACTTGCTTGGATTCCTACAAAGAAGC (SEQ ID NO: 31:“BET-025”) and 5′-GCCCTCGAGTCGACCTTGTCATCATCGTCGTTTCGGAGGTAACCTGTAAG(SEQ ID NO: 32: “BET-026”) that also incorporated a restriction enzymecleavage site (BsaI) upstream of the first codon of the IFN-beta. The 3′PCR primer (SEQ ID NO: 32: BET-026) for the IFN-beta gene eliminated theIFN-beta termination codon, and incorporated both an in frameenterokinase linker sequence (DDDDK) and a terminal restriction enzymesite (XhoI), useful for subcloning into the expression vector. The BsaIsite introduced upstream of the IFN-beta coding sequence allowed us toligate the VCAM-1 signal sequence upstream and in frame with theIFN-beta gene coding sequence. This VCAM-1 signal sequence was alsogenerated by PCR using primer pairs 5′-CAAGCTTGCTAGCGGCCGCGG-3′ (SEQ IDNO: 33: “BET-023” and 5′-GGTGGTCTCACATGGCTTGAGAAGCTGC-3′(SEQ ID NO: 34:“BET-024”) that contained a 5′ restriction enzyme cleavage site (NotI,for ligation onto the pDSW247 NotI cloning site) and a 3′ restrictionenzyme cleavage site (BsaI, for ligation onto the IFN-beta-1a 5′ PCRfragment). The template for PCR was the human vascular cell adhesionmolecule-1 (VCAM-1) cDNA (GenBank accession number X53051).

[0158] To create the IFN-beta-1a/Fc fusion gene the following procedureswere performed. The murine IgG2a fragment was removed from pEAG293 bygel purification of a SalI+BamHI digestion DNA fragment. PlasmsidpEAG293 is a Bluescript IISK+(Stratagene, LaJolla Calif., catalogue#212205) subclone of the hinge, CH2 and CH3 domains of murine IgG2a(GenBank accession number V00798). The PCR primer pairs5′-AGGTSMARCTGCAGSAGTCW-3′ (SEQ ID NO: 35), where S=C or G, M=A or C,R=A or G, W=A or T, and 5′-CTGAGCTCATTTACCCGGAGTCCGGGA GAAGCTCTT-3′ (SEQID NO: 36) created flanking SalI and NotI sites at the 5′ and 3′ ends ofthe cassette, respectively. The murine IgG2a Fc domain cassette differsfrom the GenBank sequence at a single base (codon V369), creating asilent mutation. Hence, wild type Fc protein is expressed from thisIgG2a Fc cassette.

[0159] The DNA fragment containing the VCAM-1 signal sequence fused tothe huIFN-beta gene with the C-terminal enterokinase linker sequence,was excised from pCMG258 by a NotI to BamHI digestion and gel purified.The SalI site was present on the original pDSW247 plasmid, and islocated immediately downstream and in frame with the IFN-beta genecoding sequence. The plasmid vector pDSW247 was prepared as a gelpurified NotI+BamHI fragment (see Example 1). A 3-way ligation wasperformed, using the above mentioned fragments, to assemble the finalexpression vector encoding the IFN-beta-1a/IgG2a fusion. This expressionplasmid was named pCMG261 and contains the VCAM-1 signal sequence in afusion with the gene for mature human IFN-beta, enterokinase linkersequence and murine IgG2a Fc domain. The full DNA (SEQ ID NO:1) andprotein sequence (SEQ ID NO:2) of the fusion protein are shown in FIG.2.

EXAMPLE 3 Production of Interferon-beta-1a Fusion Protein in MammalianCells

[0160] The recombinant IFN-beta/Fc expression vector, pCMG261 wastransiently tranfected into human EBNA 293 kidney cells to achieveexpression of an IFN-beta-1a fusion protein of the invention. Thisrecombinant expression plasmid is transfected by the lipofectamineprotocol (catalogue # 18324-020, Life Techonologies) in EBNA 283 humankidney cells according to the protocol of the manufacturer (LifeTechnologies, Gaithersburg, Md., Hawley-Nelson, P., Ciccarone, V.,Gebeyehu, G. Jessee, J., Felgner, P. L. (1993) Focus 15.73) using 1-3micrograms plasmid DNA for a 100 mm culture dish of EBNA 293 cells. Onthe the day following lipofectamine transfection of cells, the media isreplaced with growth media (Dulbecco's modified Eagle's medium, 10%fetal bovine serum, 4 mM glutamine, 250 micogram Gentecin/ml (LifeTechnologies, Gaithersburg, Md.). The conditioned media is harvested 3-4days later and the concentration of IFN-beta-1a-Fc was determined asdescribed below.

[0161] Production of a IFN-beta/Fc fusion protein in other mammaliancell and prokaryotic cell expression systems could also be performedupon transfer of the protein coding region for the fusion protein intoappropriate expression vectors for those systems. Alternative expressionsystems would include mammalian cell expression systems such as chinesehamster ovary (CHO) cells (Barsoum, J. (1995, Methods in Mol. Biol. 48,chapter 18, 225-237) and NS-0 murine cells (Rossman, C. et al. 1996,Protein Expression and Pur. 7, 335-342), and COS7 green monkey kidneycells (Ettinger, R. et. al. 1996, Proc. Natil. Acad. Sci. USA, 93:23,13102-13107). Other eukaryotic expression systems that would beapplicable would be the yeast Pichia pastoris (Eldin, P. E. et al. 1997,J. Immun. Methods, 201, 67-75) and Saccharomyces cerevisiae (Horwitz, A.H., 1988, Proc. Natil. Acad. Sci. USA, 85, 8678-8682).

[0162] Quantitation of the IFN-beta-1a-Fc protein expression levels inthe culture supernatants from transfected EBNA 293 cells was performedby ELISA using a protein A purified IgG fraction of rabbitanti-IFN-beta-1a polyclonal antibodies (the antigen was purifiedIFN-beta-1a, Biogen, Inc.) to coat 96-well plates. The antibody detectsIFN-beta-1a standards and culture supernatants in an interferonconcentration range between 10 ng/mL and 0.3 ng/mL. Biotinylated rabbitpolyclonal anti-IFN-beta-1a (same antibodies as above) andstreptavidin-linked horseradish peroxidase were used to detect boundinterferons. To confirm ELISA values, western blot analysis wasperformed in which reduced culture supernatants and IFN-beta-1astandards were run on 5-20% Tris-glycine gels (Novex, San Diego,Calif.), transferred to PVDF membrane (Amersham Life Science, Inc.,Cleveland, Ohio) and detected with a different rabbit polyclonal serum(raised against IFN-beta-1a), followed by horseradish peroxidaselinked-donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, Pa.)antibodies.

EXAMPLE 4 Antiviral Activity of IFN-beta-1a/Murine IgG2a Fusion Protein

[0163] Human lung carcinoma cells (A549) were pretreated for 24 hourswith IFN-beta-1a or IFN-beta-murine IgG2a (61, 41, 27, 18, 12, 8.2, 5.5,3.7, 2.5, 1.6 pg/mL) prior to challenge with encephalomyocarditis virus(EMCV). Following a two-day incubation with the virus, viable cells werestained with a solution of XTT:PMS(2,3-bis(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilideinner salt:Phenazine methosulfate, at 333 μg/mL and 2 ng/mL,respectively, in phosphate buffered saline) and detected by spectroscopyat 450 nM. The assay was performed using triplicate data points for eachIFN concentration.

[0164] In FIG. 8 the standard deviations are shown as error bars. The50% cytopathic effect for IFN-beta-1a was determined to be approximately0.4 pM. The 50% cytopathic effect for IFN-beta-murine IgG2a was found tobe 0.15 pM.

EXAMPLE 5 Construction and Production of a Human InterferonBeta-1a/Human IgG1 Fc Fusion Protein

[0165] A. Construction of Human Interferon Beta-1a/Human IgG1 Fc FusionProtein

[0166] PCR technology was employed to create an expression plasmidencoding the human IFN beta DNA sequence fused to the Fc portion (hinge,CH2 and CH3 domains) of the human IgG1 heavy chain molecule.

[0167] EBNA construct: The plasmid vector pCH269 is a derivative ofpCEP4 (Invitrogen, Carlsbad, Calif.) from which the EBNA-1 gene has beendeleted. The plasmid was used for the construction of an expressionvector useful for transient protein expression in EBNA 293 human kidneycells (Invitrogen, Carlsbad, Calif.; Shen E. S., et.al 1995, Gene 156,235-239).

[0168] The fusion protein expression cassette was assembled from threeDNA fragments: a Not I/Sal I fragment encoding the VCAM-1 signalsequence in frame and fused to the sequence encoding human IFN beta, aSal I/Not I fragment encoding the hinge, CH2 and CH3 domains of humanIgG1, and a Not I fragment of EBNA expression vector pCH269.

[0169] Two distinct Not I/Sal I fragments encoding the mature VCAM-1signal sequence in frame and fused to the human IFN beta gene were madeby PCR technology. The PCR template was plasmid pCMG258 (see Example 2above) which encodes the mature VCAM-1 signal sequence in frame andfused to the human IFN beta gene, which itself is in frame and fused tothe enterokinase linker sequence. Two sets of PCR primers were used. Oneset of primers (5′-AGCTTGCTAGCGGCCGCGGCCTCACTGGCTTCA-3′ (SEQ ID NO: 37),and 5′-ATACGCGTCGACGTTTCGGAGGTAACATGTAAGTCTG-3′: (SEQ ID NO: 38))introduced an amino acid change from G to C at position 162. Thisfragment is called human IFN beta-C162.

[0170] The second primer set(5′-AGCTTGCTAGCGGCCGCGGCCTCACTGGCTTCA-3′(SEQ ID NO: 39), and5′-TACACGTCGACGCTGCCACCACCGCCGTTTCGGAGGTAACATGTAAGTCTG-3′: SEQ ID NO:40)) also introduced the G162 to C162 amino acid substitution andchanged the enterokinase linker sequence (DDDDK) to a GGGGS linkersequence in frame and fused 3′ to the human IFN beta gene. This fragmentis called human IFN beta-C162/G4S. Both sets of primers contain a 5′ NotI site to enable ligation into pCH269, and a 3′ Sal I cleavage site toenable ligation with the Sal I/Not I fragment of human IgG1.

[0171] The human IgG1 fragment which encodes the hinge, CH2 and CH3domains of human IgG1 was prepared by restriction enzyme (Sal I/Not I)digestion of plasmid pEAG409, a derivative of plasmid SAB 144 (describedin U.S. Pat. No. 5,547,853). The fragment was excised and gel purified.The EBNA expression vector plasmid pCH269 was digested with Not I andgel purified.

[0172] Two human IFN beta-human IgG1 Fc fusion constructs were generatedby two three-way ligations. One construct, called ZL6206 contains theG4S linker; the other construct, called ZL5107, is a direct fusion. Thefull DNA and protein sequences of the open reading frames of the directfusion (see FIG. 10) are shown in SEQ ID NO: 41 and SEQ ID NO: 42,respectively. The full DNA and protein sequences of the open readingframes of the linker fusion (see FIG. 11) are shown in SEQ ID NO: 43 andSEQ ID NO: 44, respectively.

[0173] CHO Construct:

[0174] A human IFN beta-human IgG1 Fc fusion CHO stable expressionconstruct was made which was comprised of the human IFN beta directlylinked to human IgG1 Fc. The human IFN beta-human IgG1 Fc fragment wascut from plasmid ZL5107 with Not I and gel purified; it was ligated intothe Not I site of pEAG347 (an expression vector containing tandem SV40early and Adenovirus major late promoters [derived from pAD2betaplasmid], a unique NotI cloning site, followed by SV40 latetranscription termination and polyA signals [derived from pCMVbetaplasmid]. pEAG347 contains a pUC19-derived plasmid backbone and apSV2dhfr-derived dhfr for MTX selection and amplification in transfectedCHO cells.).

[0175] B. Production of Human Interferon-Beta-1a/Human IgG1 Fc FusionProtein in Mammalian Cells

[0176] Transient Transfection of Human IFN Beta Fusion Constructs intoEBNA293 Cells:

[0177] The recombinant IFN-beta/human IgG1 Fc expression vectorsdescribed above were transiently transfected into human EBNA 293 kidneycells to achieve expression of an IFN-beta-1a fusion protein of theinvention. These recombinant expression plasmids were transfected by thelipofectamine protocol (catalogue#18324-020, Life Technologies) in EBNA293 human kidney cells according to the protocol described in Example 3above.

[0178] Stable Transfection of Human IFN Beta-1a/Human IgG1 Fc FusionConstruct (no Linker) into dhfr-CHO Cells:

[0179] The recombinant IFN-beta/human IgG1 Fc (with no linker) dhfrcontaining expression vector described above was stably transfected intodhfr-CHO cells to achieve expression of an IFN-beta-1a fusion protein ofthe invention. This recombinant expression plasmid was transfected byelectroporation and selection of positive clones was accomplishedaccording the following protocol:

[0180] Plasmid DNA (20 mcg) digested with Bgl II was precipitated,resuspended in 800 mcl of HEPES buffer and added to 10×10⁷ CHO cells/ml.Following electroporation, cells were cultured in DMEM complete mediafor 2 days. Cells were then split into 20-40 10 cm dishes with completeDMEM/dialyzed 10% FBS and cultured for 5 days before moving the cellsinto selection media with escalating (50-200 ng/ml) concentrations ofMTX in DMEM for two weeks. At the end of two weeks, single colonies ofcells were selected and expanded. Supernatants derived from 22 CHOclones were tested in antiviral assays.

[0181] Activity:

[0182] The anti-viral activity of the fusion proteins was determined inCPE assays as described in Example 4. Based on the 60 MU/mg specificactivity of the interferon-beta-1a standard used in the assay, theactivity of the transiently (EBNA) expressed humaninterferon-beta-1a/human IgG1 Fc fusion protein with the linker was 900U/ml and the activity without a linker was 440 U/ml. The activity of CHOexpressed human interferon-beta-1a/human IgG1 Fc fusion protein was 50U/ml.

EXAMPLE 6 Measurement of Interferon-beta-1a Antiviral Activity in thePlasma of Mice Treated with Interferon-beta-1a andInterferon-beta-1a/Murine IgG2a Fusion Protein

[0183] Mice (C57/B16) are injected i.v. through the tail vein with50,000 Units of interferon-beta-1a (bulk) or 5,000 Units ofinterferon-beta-1a-murine IgG2a fusion protein. An equal volume ofphosphate buffer is given as a control.

[0184] Blood is sampled through retro-orbital bleeds at different timepoints (immediately, 0.25, 1, 4, 24 and 48 hours) after interferon betainjection. There are at least 3 mice per time point. Whole blood iscollected into tubes containing anticoagulant, cells are removed and theresulting plasma frozen until time of assay. The plasma samples arediluted 1:10 into serum free assay media and passed through a 0.2 umsyringe filter.

[0185] The diluted samples are then titrated into designated wells of a96 well tissue culture plate containing A549 cells. A standardInterferon-beta-1a (10, 6.7, 4.4, 2.9, 1.3, 0.9 and 0.6 U/ml AVONEX) and4 samples were run on every plate. The cells are pretreated with samplesfor 24 hours prior to challenge with EMC virus. Following a two-dayincubation with virus, viable cells are stained with a solution of MTT(at 5 mg/ml in phosphate buffer) for 1 hour, washed with phosphatebuffer, and solubilized with 1.2 N HCl in isopropanol. The wells wereread at 450 nm. Standard curves are generated for each plate and used todetermine the amount of interferon-beta-1a activity in each sample. Theactivity in the samples from the different mice are graphed against thetime points in FIG. 9.

[0186] The slower loss of interferon-beta-1a fusion from circulation asa function of time indicates that the half life of the fusion proteinsample is much longer than that of the unmodified interferon-beta-1acontrol. A second highly significant finding from the study was thatvery little of the fusion protein was lost during the distributionphase, as evidenced by the similar high levels of activity at the 15 and60 minutes timepoints. The data indicate that, unlike the controlinterferon-beta-1a, the distribution of the interferon-beta-1a fusionprotein is largely limited to the vasculature.

EXAMPLE 7 Comparative Pharmacokinetics and Pharmacodynamics in Primates

[0187] Comparative studies are conducted with interferon-beta 1a fusionand native interferon-beta 1a (as non formulated bulk intermediateAVONEX® interferon-beta-1a in 100 mM sodium phosphate, 200 mM NaCl, pH7.2) to determine their relative stability and activity in primates. Inthese studies, the pharmacokinetics and pharmacodynamics of theinterferon-beta-1a fusion in primates is compared to that of nativeinterferon-beta 1a and reasonable inferences can be extended to humans.

[0188] Animals and Methods

[0189] Study Design

[0190] This is a parallel group, repeat dose study to evaluate thecomparative pharmacokinetics and pharmacodynamics of interferon-beta-1afusion protein and nonfusion interferon-beta-1a.

[0191] Healthy primates (preferably rhesus monkeys) are used for thisstudy. Prior to dosing, all animals will be evaluated for signs of illhealth by a Lab Animal Veterinary on two occasions within 14 days priorto test article administration; one evaluation must be within 24 hoursprior to the first test article administration. Only healthy animalswill receive the test article. Evaluations will include a generalphysical examination and pre-dose blood draws for baseline clinicalpathology and baseline antibody level to interferon-beta-1a. All animalswill be weighed and body temperatures will be recorded within 24 hoursprior to test article administrations.

[0192] Twelve subjects are enrolled and assigned to groups of three toreceive 1 MU/kg of interferon-beta-1a as either a fused or a non-fused,but otherwise identical interferon-beta-1a. Administration is by eitherthe subcutaneous (SC) or intravenous (IV) routes. Six male animals willreceive test article by the IV route (3/treatment) and another 6 maleanimals will receive test article by the SC route (3/treatment). Allanimals must be naive to interferon-beta treatment. Each animal will bedosed on two occasions; doses will be separated by four weeks. The dosevolume will be 1.0 mL/kg.

[0193] Blood is drawn for pharmacokinetic testing at 0, 0.083, 0.25,0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 48, 72, and 96 hours following eachinjection. Blood samples for measurements of the interferon inducedbiological response marker, serum neopterin, are drawn at 0, 24, 48, 72,96, 168, 336, 504 hours following administration of study drug.

[0194] Evaluations during the study period include clinical observationsperformed 30 minutes and 1 hour post-dose for signs of toxicitiy. Dailycageside observations will be performed and general appearance, signs oftoxicity, discomfort, and changes in behavior will be recorded. Bodyweights and body temperatures will be recorded at regular intervalsthrough 21 days post-dose.

[0195] Assay Methods

[0196] Levels of interferon beta in serum are quantitated using acytopathic effect (CPE) bioassay. The CPE assay measures levels ofinterferon-mediated antiviral activity. The level of antiviral activityin a sample reflects the number of molecules of active interferoncontained in that sample at the time the blood is drawn. This approachhas been the standard method to assess the phannacokinetics ofinterferon beta. The CPE assay used in the current study detects theability of interferon beta to protect human lung carcinoma cells (A549,#CCL-185, ATCC, Rockville, Md.) from cytotoxicity due toencephalomyocarditis (EMC) virus. The cells are preincubated for 15 to20 hours with serum samples to allow the induction and synthesis ofinterferon inducible proteins that then mount an antiviral response.Afterwards EMC virus is added and incubated for a further 30 hoursbefore assessment of cytotoxicity is made using a crystal violet stain.An internal interferon beta standard as well as an interferon-beta-Iginternal standard is tested concurrently with samples on each assayplate. This standard is calibrated against a natural human fibroblastinterferon reference standard (WHO Second International Standard forInterferon, Human Fibroblast, Gb-23-902-53). Each assay plate alsoincludes cell growth control wells containing neither interferon beta ofany kind nor EMC, and virus control wells contain cells and EMC but nointerferon beta. Control plates containing the standard and samples arealso prepared to determine the effect, if any, of the samples on cellgrowth. These plates are stained without the addition of virus.

[0197] Samples and standards are tested in duplicate on each of tworeplicate assay plates, yielding four data points per sample. Thegeometric mean concentration of the four replicates is reported. Thelimit of detection in this assay is 10 units (U)/ml.

[0198] Serum concentrations of neopterin are determined at the clinicalpharmacology unit using commercially available assays.

[0199] Pharmacokinetic and Statistical Methods

[0200] RstripTM software (MicroMath, Inc., Salt Lake City, Utah) is usedto fit data to pharmacokinetic models. Geometric mean concentrations areplotted by time for each group. Since assay results are expressed indilutions, geometric means are considered more appropriate thanarithmetic means. Serum interferon levels are adjusted for baselinevalues and non-detectable serum concentrations are set to 5 U/ml, whichrepresents one-half the lower limit of detection.

[0201] For IV infusion data, a two compartment IV infusion model is fitto the detectable serum concentrations for each subject, and the SC dataare fit to a two compartment injection model.

[0202] The following pharmacokinetic parameters are calculated:

[0203] (i) observed peak concentration, C_(max) (U/ml);

[0204] (ii) area under the curve from 0 to 48 hours, AUC using thetrapezoidal rule;

[0205] (iii) elimination half-life; and, from IV infusion data (if IV isemployed):

[0206] (iv) distribution half-life (h);

[0207] (v) clearance (ml/h)

[0208] (vi) apparent volume of distribution, Vd (L).

[0209] WinNonlin (Version 1.0, Scientific Consulting Inc., Apex, N.C.)software is used to calculate the elimination half-lives after SC and IMinjection.

[0210] For neopterin, arithmetic means by time are presented for eachgroup. E_(max), the maximum change from baseline, is calculated.C_(max), AUC and E_(max) are submitted to a one-way analysis of varianceto compare dosing groups. C_(max) and AUC are logarithmicallytransformed prior to analysis; geometric means are reported.

EXAMPLE 8 Anti-Angiogenic Effects of Interferon Beta-1a Fusion

[0211] Assessment of the Ability of an Interferon-beta-1a Fusion toInhibit Endothelial Cell Proliferation in Vitro

[0212] Human venous endothelial cells (Cell Systems, Cat. # 2V0-P75) andhuman dermal microvascular endothelial cells (Cell Systems, Cat. #2M1-C25) are maintained in culture with CS-C Medium Kit (Cell Systems,Cat. # 4Z0-500). Twenty-four hours prior to the experiment, cells aretrypsinized, and resuspended in assay medium, 90% M199 and 10% fetalbovine serum (FBS), and are adjusted to desired cell density. Cells arethen plated onto gelatin-coated 24 or 96 well plates, either at 12,500cells/well or 2,000 cells/well, respectively.

[0213] After overnight incubation, the assay medium is replaced withfresh medium containing 20 ng/ml of human recombinant basic FibroblastGrowth Factor (Becton Dickinson, Cat. # 40060) and variousconcentrations of fusion and non-fusion interferon-beta-1a proteins orpositive control (endostatin can be used as a positive control, as couldan antibody to bFGF). The final volume is adjusted to 0.5 ml in the 24well plate or 0.2 ml in the 96 well plate.

[0214] After seventy-two hours, cells are trypsinized for Coultercounting, frozen for CyQuant fluorescense reading, or labeled with [3H]thymidine.

[0215] This in vitro assay tests the human interferon-beta molecules ofthe invention for effects on endothelial cell proliferation which may beindicative of anti-angiogenic effects in vivo. See O'Reilly, M. S., T.Boehm, Y. Shing, N. Fukal, G. Vasios, W. Lane, E. Flynn, J. Birkhead, B.Olsen, and J. Folkman. (1997). Endostatin: An Endogenous Inhibitor ofAngiogensis and Tumor Growth. Cell 88, 277-285.

EXAMPLE 9 In Vivo Model to Test Anti-Angiogenic and NeovascularizationEffects of Interferon-beta-1a/Ig Fusion

[0216] A variety of models have been developed to test for theanti-angiogenic and anti-neovascularization effects of the moleculesdescribed herein. Some of these models have been described in U.S. Pat.No. 5,733,876 (Mar. 31,1998: “Method of inhibiting angiogenesis) andU.S. Pat. No. 5,135,919 (Aug. 4, 1992:” Method and a pharmaceuticalcomposition for the inhibition of angiogenesis ”). Other assays includethe shell-less chorioallantoic membrane (CAM) assay of S. Taylor and J.Folkman; Nature, 297, 307 (1982) and R. Crum. S. Szabo and J. Folkman;Science. 230. 1375 (1985); the mouse dorsal air sac methodantigiogenesis model of Folkman, J. et al.; J. Exp. Med., 133, 275(1971) and the rat corneal micropocket assay of Gimbrone, M. A. Jr. etal., J. Natl. Cancer Inst. 52, 413(1974) in which cornealvascularization is induced in adult male rats of the Sprague-Dawleystrain (Charles River, Japan) by implanting 500 ng of basic FGF (bovine,R & D Systems, Inc.) impregnated in EVA (ethylene-vinyl acetatecopolymer) pellets in each cornea.

[0217] Other methods for testing interferon-beta/Ig fusions foranti-angiogenic effects in an animal model include (but are not limitedto) protocols for screening new potential anticancer agents as describedin the original Cancer Chemotherapy Reports, Part 3, Vol. 3, No.2,September 1972 and the supplement In Vivo Cancer Models, 1976-1982, NIHPublication No. 84-2635, February 1984. Because of the species barriersof Type I interferons, to assess the anti-angiogenic activity ofinterferon-beta fusions in rodent models, rodent interferon-beta/:Igfusion preparations are generated. Such screening methods areexemplified by a protocol to test for the anti-angiogenic effects ofmurine interferon-beta/Ig fusions on subcutaneously-implanted Lewis LungCarcinoma.

[0218] Origin of Tumor Line:

[0219] Arose spontaneously in 1951 as a carcinoma of the lung in aC57BL/6 mouse.

[0220] Summary of Test Procedures:

[0221] A tumor fragment is implanted subcutaneously in the axillaryregion of a B6D2F1 mouse. The test agent (i.e, a fusion protein of theinvention) is administered at various doses, subcutaneously (SC) orintraperitoneally (IP) on multiple days following tumor implantation.The parameter measured is median survival time. Results are expressed asa percentage of control survival time.

[0222] Animals:

[0223] Propagation: C57BL/6 mice.

[0224] Testing: B6D2F1 mice.

[0225] Weight: Mice should be within a 3 gm weight range with a minimumweight of 18 gm for males and 17 gm for females.

[0226] Sex: One sex is used for all test and control animals in oneexperiment.

[0227] Source: One source, if feasible, for all animals in oneexperiment.

[0228] Experiment Size:

[0229] Ten animals per test group.

[0230] Tumor Transfer:

[0231] Propagation:

[0232] Fragment: Prepare a 2-4 mm fragment of a s.c. donor tumor

[0233] Time: Day 13-15

[0234] Site: Implant the fragment s.c. in the axillary region with apuncture in the inguinal region.

[0235] Testing:

[0236] Fragment: Prepare a 2-4 mm fragment of s.c. donor tumor.

[0237] Time: Day 13-15.

[0238] Site: Implant the fragment s.c. in the axillary region with apuncture in the inguinal region.

[0239] Testing Schedule:

[0240] Day 0: Implant tumor. Run bacterial cultures. Test positivecontrol compound in every odd-numbered experiment. Prepare materials.Record deaths daily.

[0241] Day 1: Check cultures. Discard experiment if contaminated.Randomize animals. Treat as instructed (on day 1 and on following days).

[0242] Day 2: Recheck cultures. Discard experiment if contaminated.

[0243] Day 5: Weigh Day 2 and day of initial test agent toxicityevaluation.

[0244] Day 14: Control early-death day.

[0245] Day 48: Control no-take day.

[0246] Day 60: End and evaluate experiment. Examine lungs grossly fortumor.

[0247] Quality Control:

[0248] Schedule the positive control compound (NSC 26271 (Cytoxan at adose of 100 mg/kg/injection)) in every odd-numbered experiment, theregimen for which is intraperitoneal on Day 1 only. The lowerTest/Control limit for the positive control is 140%. The acceptableuntreated control median survival time is 19-35.6 days.

[0249] Evaluation:

[0250] The parameter measured is median survival time Compute meananimal body weights for Day 1 and Day 5, compute Test/Control ratio forall test groups with. The mean animal body weights for staging day andfinal evaluation day are computed. The Test/Control ratio is computedfor all test groups with >65% survivors on Day 5. A Test/Control ratiovalue <86% indicates toxicity. An excessive body weight changedifference (test minus control) may also be used in evaluating toxicity.

[0251] Criteria for Activity:

[0252] An initial Test/Control ratio greater than or equal to 140% isconsidered necessary to demonstrate moderate activity. A reproducibleTest/Control ratio value of greater than or equal to 150% is consideredsignificant activity.

1 44 1 1197 DNA murine 1 atgagctaca acttgcttgg attcctacaa agaagcagcaattttcagtg tcagaagctc 60 ctgtggcaat tgaatgggag gcttgaatac tgcctcaaggacaggatgaa ctttgacatc 120 cctgaggaga ttaagcagct gcagcagttc cagaaggaggacgccgcatt gaccatctat 180 gagatgctcc agaacatctt tgctattttc agacaagattcatctagcac tggctggaat 240 gagactattg ttgagaacct cctggctaat gtctatcatcagataaacca tctgaagaca 300 gtcctggaag aaaaactgga gaaagaagat ttcaccaggggaaaactcat gagcagtctg 360 cacctgaaaa gatattatgg gaggattctg cattacctgaaggccaagga gtacagtcac 420 tgtgcctgga ccatagtcag agtggaaatc ctaaggaacttttacttcat taacagactt 480 acaggttacc tccgaaacga cgatgatgac aaggtcgacaaaactcacac atgcccaccg 540 tgcccagcac ctgaactcct ggggggaccg tcagtcttcctcttcccccc aaaacccaag 600 gacaccctca tgatctcccg gacccctgag gtcacatgcgtggtggtgga cgtgagccac 660 gaagaccctg aggtcaagtt caactggtac gtggacggcgtggaggtgca taatgccaag 720 acaaagccgc gggaggagca gtacaacagc acgtaccgtgtggtcagcgt cctcaccgtc 780 ctgcaccagg actggctgaa tggcaaggag tacaagtgcaaggtctccaa caaagccctc 840 ccagccccca tcgagaaaac catctccaaa gccaaagggcagccccgaga accacaggtg 900 tacaccctgc ccccatcccg ggatgagctg accaagaaccaggtcagcct gacctgcctg 960 gtcaaaggct tctatcccag cgacatcgcc gtggagtgggagagcaatgg gcagccggag 1020 aacaactaca agaccacgcc tcccgtgttg gactccgacggctccttctt cctctacagc 1080 aagctcaccg tggacaagag caggtggcag caggggaacgtcttctcatg ctccgtgatg 1140 catgaggctc tgcacaacca ctacacgcag aagagcctctccctgtctcc cgggaaa 1197 2 399 PRT murine 2 Met Ser Tyr Asn Leu Leu GlyPhe Leu Gln Arg Ser Ser Asn Phe Gln 1 5 10 15 Cys Gln Lys Leu Leu TrpGln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30 Lys Asp Arg Met Asn PheAsp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu AspAla Ala Leu Thr Ile Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile PheArg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Val GluAsn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His Leu Lys Thr ValLeu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110 Arg Gly Lys LeuMet Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115 120 125 Ile Leu HisTyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr 130 135 140 Ile ValArg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu 145 150 155 160Thr Gly Tyr Leu Arg Asn Asp Asp Asp Asp Lys Val Asp Lys Thr His 165 170175 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 180185 190 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr195 200 205 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp ProGlu 210 215 220 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His AsnAla Lys 225 230 235 240 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser 245 250 255 Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Glu Tyr Lys 260 265 270 Cys Lys Val Ser Asn Lys Ala Leu Pro AlaPro Ile Glu Lys Thr Ile 275 280 285 Ser Lys Ala Lys Gly Gln Pro Arg GluPro Gln Val Tyr Thr Leu Pro 290 295 300 Pro Ser Arg Asp Glu Leu Thr LysAsn Gln Val Ser Leu Thr Cys Leu 305 310 315 320 Val Lys Gly Phe Tyr ProSer Asp Ile Ala Val Glu Trp Glu Ser Asn 325 330 335 Gly Gln Pro Glu AsnAsn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 340 345 350 Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 355 360 365 Trp Gln GlnGly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 370 375 380 His AsnHis Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 395 3 549DNA murine 3 tccgggggcc atcatcatca tcatcatagc tccggagacg atgatgacaagatgagctac 60 aacttgcttg gattcctaca aagaagcagc aattttcagt gtcagaagctcctgtggcaa 120 ttgaatggga ggcttgaata ctgcctcaag gacaggatga actttgacatccctgaggag 180 attaagcagc tgcagcagtt ccagaaggag gacgccgcat tgaccatctatgagatgctc 240 cagaacatct ttgctatttt cagacaagat tcatctagca ctggctggaatgagactatt 300 gttgagaacc tcctggctaa tgtctatcat cagataaacc atctgaagacagtcctggaa 360 gaaaaactgg agaaagaaga tttcaccagg ggaaaactca tgagcagtctgcacctgaaa 420 agatattatg ggaggattct gcattacctg aaggccaagg agtacagtcactgtgcctgg 480 accatagtca gagtggaaat cctaaggaac ttttacttca ttaacagacttacaggttac 540 ctccgaaac 549 4 183 PRT murine 4 Ser Gly Gly His His HisHis His His Ser Ser Gly Asp Asp Asp Asp 1 5 10 15 Lys Met Ser Tyr AsnLeu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe 20 25 30 Gln Cys Gln Lys LeuLeu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys 35 40 45 Leu Lys Asp Arg MetAsn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu 50 55 60 Gln Gln Phe Gln LysGlu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu 65 70 75 80 Gln Asn Ile PheAla Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp 85 90 95 Asn Glu Thr IleVal Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile 100 105 110 Asn His LeuLys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe 115 120 125 Thr ArgGly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly 130 135 140 ArgIle Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp 145 150 155160 Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg 165170 175 Leu Thr Gly Tyr Leu Arg Asn 180 5 60 DNA Homo sapiens 5gatctagcaa tgctgcctgt gctgccctcc tggctgcctt gaatgggagg cttgaatact 60 651 DNA Homo sapiens 6 tattatggga ggattctgca ttacctgaag gccaaggagtactcacactg t 51 7 76 DNA Homo sapiens 7 aattgaatgg gagggctgca gcttgcgctgcagacaggat gaactttgac atccctgagg 60 agattaagca gctgca 76 8 51 PRT Homosapiens 8 Ala Ala Thr Thr Gly Ala Ala Thr Gly Gly Gly Ala Gly Gly CysThr 1 5 10 15 Thr Gly Ala Ala Thr Ala Cys Thr Gly Cys Cys Thr Cys AlaAla Gly 20 25 30 Gly Ala Cys Ala Gly Gly Ala Thr Gly Ala Ala Cys Thr ThrThr Gly 35 40 45 Ala Cys Ala 50 9 60 DNA Homo sapiens 9 ttctccggagacgatgatga caagatgagc tacaacttgc ttggattcct acaaagaagc 60 10 50 DNA Homosapiens 10 cgtcagagct gaaatcctag caaactttgc attcattgca agacttacag 50 1147 DNA Homo sapiens 11 ggtggtctca catgagctac aacttgcttg gattcctacaaagaagc 47 12 50 DNA Homo sapiens 12 gccctcgagt cgaccttgtc atcatcgtcgtttcggaggt aacctgtaag 50 13 21 DNA Homo sapiens 13 caagcttgct agcggccgcgg 21 14 28 DNA Homo sapiens 14 ggtggtctca catggcttga gaagctgc 28 15 20DNA Homo sapiens 15 aggtsmarct gcagsagtcw 20 16 36 DNA Homo sapiens 16ctgagctcat ttacccggag tccgggagaa gctctt 36 17 33 DNA Homo sapiens 17agcttgctag cggccgcggc ctcactggct tca 33 18 37 DNA Homo sapiens 18atacgcgtcg acgtttcgga ggtaacatgt aagtctg 37 19 33 DNA Homo sapiens 19agcttgctag cggccgcggc ctcactggct tca 33 20 51 DNA Homo sapiens 20tacacgtcga cgctgccacc accgccgttt cggaggtaac atgtaagtct g 51 21 39 DNAHomo sapiens 21 gccgctcgag ttatcagttt cggaggtaac ctgtaagtc 39 22 72 DNAHomo sapiens 22 ggaatgcttc aattgttgct gcactcctga gcaatgtcta tcatcagataaaccatctga 60 agacagttct ag 72 23 72 DNA Homo sapiens 23 ggaatgagaccattgttgag aacctcctgg ctaatgtcgc tcatcagata gcacatctgg 60 ctgcagttct ag72 24 44 DNA Homo sapiens 24 ctagctgcaa aactggctgc agctgatttc accaggggaaaact 44 25 69 DNA Homo sapiens 25 ctagaagaaa aactggagaa agaagcagctaccgctggaa aagcaatgag cgcgctgcac 60 ctgaaaaga 69 26 51 DNA Homo sapiens26 tattatggga ggattctgca ttacctgaag gccaaggagt actcacactg t 51 27 76 DNAHomo sapiens 27 catgagcagt ctgcacctga aaagatatta tggggcaatt gctgcatacctggcagccaa 60 ggagtactca cactgt 76 28 87 DNA Homo sapiens 28 catgagcagtctgcacctga aaagatatta tgggaggatt ctgcattacc tgaaggccgc 60 tgcatactcacactgtgcct ggacgat 87 29 87 DNA Homo sapiens 29 catgagcagt ctgcacctgaaaagatatta tgggaggatt ctgcattacc tgaaggcaaa 60 ggagtacgct gcatgtgcctggacgat 87 30 50 DNA Homo sapiens 30 cgtcagagct gaaatcctag caaactttgcattcattgca agacttacag 50 31 47 DNA Homo sapiens 31 ggtggtctca catgagctacaacttgcttg gattcctaca aagaagc 47 32 50 DNA Homo sapiens 32 gccctcgagtcgaccttgtc atcatcgtcg tttcggaggt aacctgtaag 50 33 21 DNA Homo sapiens 33caagcttgct agcggccgcg g 21 34 28 DNA Homo sapiens 34 ggtggtctcacatggcttga gaagctgc 28 35 20 DNA murine 35 aggtsmarct gcagsagtcw 20 3636 DNA murine 36 ctgagctcat ttacccggag tccgggagaa gctctt 36 37 33 DNAHomo sapiens 37 agcttgctag cggccgcggc ctcactggct tca 33 38 37 DNA Homosapiens 38 atacgcgtcg acgtttcgga ggtaacatgt aagtctg 37 39 33 DNA Homosapiens 39 agcttgctag cggccgcggc ctcactggct tca 33 40 51 DNA Homosapiens 40 tacacgtcga cgctgccacc accgccgttt cggaggtaac atgtaagtct g 5141 1257 DNA Homo sapiens 41 atgcctggga agatggtcgt gatccttgga gcctcaaatatactttggat aatgtttgca 60 gcttctcaag ccatgagcta caacttgctt ggattcctacaaagaagcag caattttcag 120 tgtcagaagc tcctgtggca attgaatggg aggcttgaatactgcctcaa ggacaggatg 180 aactttgaca tccctgagga gattaagcag ctgcagcagttccagaagga ggacgccgca 240 ttgaccatct atgagatgct ccagaacatc tttgctattttcagacaaga ttcatctagc 300 actggctgga atgagactat tgttgagaac ctcctggctaatgtctatca tcagataaac 360 catctgaaga cagtcctgga agaaaaactg gagaaagaagatttcaccag gggaaaactc 420 atgagcagtc tgcacctgaa aagatattat gggaggattctgcattacct gaaggccaag 480 gagtacagtc actgtgcctg gaccatagtc agagtggaaatcctaaggaa cttttacttc 540 attaacagac ttacatgtta cctccgaaac gtcgacaaaactcacacatg cccaccgtgc 600 ccagcacctg aactcctggg gggaccgtca gtcttcctcttccccccaaa acccaaggac 660 accctcatga tctcccggac ccctgaggtc acatgcgtggtggtggacgt gagccacgaa 720 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtggaggtgcataa tgccaagaca 780 aagccgcggg aggagcagta caacagcacg taccgtgtggtcagcgtcct caccgtcctg 840 caccaggact ggctgaatgg caaggagtac aagtgcaaggtctccaacaa agccctccca 900 gcccccatcg agaaaaccat ctccaaagcc aaagggcagccccgagaacc acaggtgtac 960 accctgcccc catcccggga tgagctgacc aagaaccaggtcagcctgac ctgcctggtc 1020 aaaggcttct atcccagcga catcgccgtg gagtgggagagcaatgggca gccggagaac 1080 aactacaaga ccacgcctcc cgtgttggac tccgacggctccttcttcct ctacagcaag 1140 ctcaccgtgg acaagagcag gtggcagcag gggaacgtcttctcatgctc cgtgatgcat 1200 gaggctctgc acaaccacta cacgcagaag agcctctccctgtctcccgg gaaatga 1257 42 418 PRT Homo sapiens 42 Met Pro Gly Lys MetVal Val Ile Leu Gly Ala Ser Asn Ile Leu Trp 1 5 10 15 Ile Met Phe AlaAla Ser Gln Ala Met Ser Tyr Asn Leu Leu Gly Phe 20 25 30 Leu Gln Arg SerSer Asn Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu 35 40 45 Asn Gly Arg LeuGlu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp Ile 50 55 60 Pro Glu Glu IleLys Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala 65 70 75 80 Leu Thr IleTyr Glu Met Leu Gln Asn Ile Phe Ala Ile Phe Arg Gln 85 90 95 Asp Ser SerSer Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu 100 105 110 Ala AsnVal Tyr His Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu 115 120 125 LysLeu Glu Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser Ser Leu 130 135 140His Leu Lys Arg Tyr Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys 145 150155 160 Glu Tyr Ser His Cys Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg165 170 175 Asn Phe Tyr Phe Ile Asn Arg Leu Thr Cys Tyr Leu Arg Asn ValAsp 180 185 190 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu LeuGly Gly 195 200 205 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp ThrLeu Met Ile 210 215 220 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val AspVal Ser His Glu 225 230 235 240 Asp Pro Glu Val Lys Phe Asn Trp Tyr ValAsp Gly Val Glu Val His 245 250 255 Asn Ala Lys Thr Lys Pro Arg Glu GluGln Tyr Asn Ser Thr Tyr Arg 260 265 270 Val Val Ser Val Leu Thr Val LeuHis Gln Asp Trp Leu Asn Gly Lys 275 280 285 Glu Tyr Lys Cys Lys Val SerAsn Lys Ala Leu Pro Ala Pro Ile Glu 290 295 300 Lys Thr Ile Ser Lys AlaLys Gly Gln Pro Arg Glu Pro Gln Val Tyr 305 310 315 320 Thr Leu Pro ProSer Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 325 330 335 Thr Cys LeuVal Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 340 345 350 Glu SerAsn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 355 360 365 LeuAsp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 370 375 380Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 385 390395 400 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro405 410 415 Gly Lys 43 1272 DNA Homo sapiens 43 atgcctggga agatggtcgtgatccttgga gcctcaaata tactttggat aatgtttgca 60 gcttctcaag ccatgagctacaacttgctt ggattcctac aaagaagcag caattttcag 120 tgtcagaagc tcctgtggcaattgaatggg aggcttgaat actgcctcaa ggacaggatg 180 aactttgaca tccctgaggagattaagcag ctgcagcagt tccagaagga ggacgccgca 240 ttgaccatct atgagatgctccagaacatc tttgctattt tcagacaaga ttcatctagc 300 actggctgga atgagactattgttgagaac ctcctggcta atgtctatca tcagataaac 360 catctgaaga cagtcctggaagaaaaactg gagaaagaag atttcaccag gggaaaactc 420 atgagcagtc tgcacctgaaaagatattat gggaggattc tgcattacct gaaggccaag 480 gagtacagtc actgtgcctggaccatagtc agagtggaaa tcctaaggaa cttttacttc 540 attaacagac ttacatgttacctccgaaac ggcggtggtg gcagcgtcga caaaactcac 600 acatgcccac cgtgcccagcacctgaactc ctggggggac cgtcagtctt cctcttcccc 660 ccaaaaccca aggacaccctcatgatctcc cggacccctg aggtcacatg cgtggtggtg 720 gacgtgagcc acgaagaccctgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 780 cataatgcca agacaaagccgcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 840 gtcctcaccg tcctgcaccaggactggctg aatggcaagg agtacaagtg caaggtctcc 900 aacaaagccc tcccagcccccatcgagaaa accatctcca aagccaaagg gcagccccga 960 gaaccacagg tgtacaccctgcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1020 ctgacctgcc tggtcaaaggcttctatccc agcgacatcg ccgtggagtg ggagagcaat 1080 gggcagccgg agaacaactacaagaccacg cctcccgtgt tggactccga cggctccttc 1140 ttcctctaca gcaagctcaccgtggacaag agcaggtggc agcaggggaa cgtcttctca 1200 tgctccgtga tgcatgaggctctgcacaac cactacacgc agaagagcct ctccctgtct 1260 cccgggaaat ga 1272 44423 PRT Homo sapiens 44 Met Pro Gly Lys Met Val Val Ile Leu Gly Ala SerAsn Ile Leu Trp 1 5 10 15 Ile Met Phe Ala Ala Ser Gln Ala Met Ser TyrAsn Leu Leu Gly Phe 20 25 30 Leu Gln Arg Ser Ser Asn Phe Gln Cys Gln LysLeu Leu Trp Gln Leu 35 40 45 Asn Gly Arg Leu Glu Tyr Cys Leu Lys Asp ArgMet Asn Phe Asp Ile 50 55 60 Pro Glu Glu Ile Lys Gln Leu Gln Gln Phe GlnLys Glu Asp Ala Ala 65 70 75 80 Leu Thr Ile Tyr Glu Met Leu Gln Asn IlePhe Ala Ile Phe Arg Gln 85 90 95 Asp Ser Ser Ser Thr Gly Trp Asn Glu ThrIle Val Glu Asn Leu Leu 100 105 110 Ala Asn Val Tyr His Gln Ile Asn HisLeu Lys Thr Val Leu Glu Glu 115 120 125 Lys Leu Glu Lys Glu Asp Phe ThrArg Gly Lys Leu Met Ser Ser Leu 130 135 140 His Leu Lys Arg Tyr Tyr GlyArg Ile Leu His Tyr Leu Lys Ala Lys 145 150 155 160 Glu Tyr Ser His CysAla Trp Thr Ile Val Arg Val Glu Ile Leu Arg 165 170 175 Asn Phe Tyr PheIle Asn Arg Leu Thr Cys Tyr Leu Arg Asn Gly Gly 180 185 190 Gly Gly SerVal Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 195 200 205 Glu LeuLeu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 210 215 220 AspThr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 225 230 235240 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 245250 255 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr260 265 270 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His GlnAsp 275 280 285 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn LysAla Leu 290 295 300 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln Pro Arg 305 310 315 320 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser ArgAsp Glu Leu Thr Lys 325 330 335 Asn Gln Val Ser Leu Thr Cys Leu Val LysGly Phe Tyr Pro Ser Asp 340 345 350 Ile Ala Val Glu Trp Glu Ser Asn GlyGln Pro Glu Asn Asn Tyr Lys 355 360 365 Thr Thr Pro Pro Val Leu Asp SerAsp Gly Ser Phe Phe Leu Tyr Ser 370 375 380 Lys Leu Thr Val Asp Lys SerArg Trp Gln Gln Gly Asn Val Phe Ser 385 390 395 400 Cys Ser Val Met HisGlu Ala Leu His Asn His Tyr Thr Gln Lys Ser 405 410 415 Leu Ser Leu SerPro Gly Lys 420

What is claimed is:
 1. An isolated polypeptide having the amino acidsequence X—Y—Z, wherein X is a polypeptide having the amino acidsequence, or portion thereof, comprising the amino acid sequence of aglycosylated interferon-beta; Y is an optional linker moiety; and Z is apolypeptide comprising at least a portion of a polypeptide other thanglycosylated interferon-beta.
 2. The isolated polypeptide of claim 1,wherein X is interferon-beta-1a.
 3. The isolated polypeptide of claim 1,wherein X is a mutant having at least one of the following properties:(a) the mutant has a higher antiviral activity than wild type interferonbeta 1a, wherein the antiviral activity is measured by viral inducedlysis of cells; (b) the mutant has, relative to wild typeinterferon-beta-1a, greater antiviral activity than antiproliferativeactivity; (c) the mutant binds interferon receptor but has, whencompared to wild type interferon-beta-1a, lowered antiviral activity andlowered antiproliferative activity relative to its receptor bindingactivity.
 4. The isolated polypeptide of claim 2, wherein the interferonbeta-1a is derivatized.
 5. The isolated polypeptide of claim 4, whereinthe derivative is a polyalkylglycol polymer.
 6. The isolated polypeptideof claim 1, wherein Z is at least a portion of a constant region. of animmunoglobulin.
 7. The isolated polypeptide of claim 6, wherein said atleast a portion of the constant region is derived from an immunoglobulinof the class selected from classes IgM, IgG, IgD, IgA, and IgE.
 8. Theisolated polypeptide of claim 7, wherein the class is IgG.
 9. Theisolated polypeptide of claim 6, wherein the at least a portion of theconstant region comprises at least a hinge, CH2 and CH3 domains.
 10. Afusion protein having an amino terminal region consisting of the aminoacid sequence of a glycosylated interferon-beta or a portion thereof andhaving a carboxy terminal region comprising at least a portion of aprotein other than glycosylated interferon-beta.
 11. The isolatedprotein of claim 10, wherein X is interferon-beta-1a.
 12. The isolatedprotein of claim 10, wherein X is a mutant having at least one of thefollowing properties: (a) the mutant has a higher antiviral activitythan wild type inteferon beta 1a, wherein the antiviral activity ismeasured by viral induced lysis of cells; (b) the mutant has, relativeto wild type interferon-beta-1a, greater antiviral activity thanantiproliferative activity; (c) the mutant binds interferon receptor buthas, compared to wild type interferon-beta-1a, lowered antiviralactivity and lowered antiproliferative activity relative to its receptorbinding activity.
 13. The isolated protein of claim 11, wherein theinterferon-beta-1a is derivatized.
 14. The isolated protein of claim 13,wherein the derivative is a polyalkylglycol polymer.
 15. The isolatedprotein of claim 10, wherein the at least a portion of the protein otherthan interferon beta is at least a portion of a constant region of animmunoglobulin.
 16. The isolated protein of claim 15, wherein said atleast a portion of the constant region is derived from an immunoglobulinof the class selected from classes IgM, IgG, IgD, IgA, and IgE.
 17. Theisolated protein of claim 16, wherein the class is IgG.
 18. The isolatedprotein of claim 15, wherein the at least a portion of the constantregion is comprises at least a hinge, CH2 and CH3 domains.
 19. Anisolated DNA sequence encoding for the protein of claims 1 and
 10. 20. Arecombinant DNA comprising the DNA sequence of claim 19 and anexpression control sequence, wherein the expression control sequence isoperatively linked to the DNA.
 21. A host cell transformed with therecombinant DNA sequence of claim
 20. 22. A method of producing arecombinant polypeptide comprising: (a) providing a population of hostcells according to claim 21; (b) growing said population of cells underconditions whereby the polypeptide encoded by said recombinant DNA isexpressed; and (c) isolating the expressed polypeptide.
 23. Aninterferon-beta fusion protein comprising a glycosylated interferon betaand additional polypeptide with which it is not natively associated, insubstantially purified form
 24. The fusion protein of claim 23, whereinsaid interferon beta is human interferon-beta-1a.
 25. The fusion proteinof claim 24, wherein said fusion has an antiviral activity that isselected from the group consisting of: (a) a higher antiviral activitythan wild type inteferon beta 1a, wherein the antiviral activity ismeasured by viral induced lysis of cells, (b) a greater antiviralactivity than antiproliferative activity, relative to wild typeinterferon-beta-1a; (c) an activity that includes receptor bindingactivity but, compared to wild type interferon-beta-1a, a loweredantiviral activity and lowered antiproliferative activity relative tosaid receptor binding activity.
 26. A pharmaceutical compositioncomprising a therapeutically effective amount of the interferon betafusion protein of claims 1, 10 and
 23. 27. A method of inhibitingangiogenesis in a subject, comprising administering to a subject aneffective amount of the composition of claim
 26. 28. The isolatedpolypeptide of claim 3, wherein the mutant is derivatized.
 29. Theisolated polypeptide of claim 27, wherein the derivative is apolyalklyglycol polymer.
 30. The isolated protein of claim 12, whereinthe mutant is derivatized.
 31. The isolated protein of claim 29, whereinthe derivative is a polyalkylglycol polymer.